Composition and method for activating latent human immunodeficiency virus (HIV)

ABSTRACT

Provided are compositions and methods for activating latent Human Immunodeficiency Virus (HIV). The compositions and methods may utilize a recombinant peptide that has a DNA binding zinc finger domain specific to the HIV long terminal repeat (LTR) sequence. The recombinant peptide may also have a transcription factor (e.g. a transcription activator) that is conjugated to the zinc finger domain. Also provided are methods of treating HIV in a subject in need of the treatment. The method may involve activation of latent HIV in cells of the subject and selectively removing such cells from the subject, providing complete and effective treatment of HIV.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a national stage entry, filed under 35 U.S.C. 371,of International Application No. PCT/US2018/056943, filed Oct. 22, 2018,which claims the benefit of U.S. Provisional Application No. 62/575,307,filed Oct. 20, 2017, the contents of which are incorporated herein byreference in their entirety and for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under P01 AI099783-01and RO1 AI111139-01 awarded by National Institute of Allergy andInfectious Disease (NIH NIAID). The government has certain rights in theinvention.

SEQUENCE LISTING

The Sequence Listing written in file048440-682001WO_Sequence_Listing_ST25.txt, created on Oct. 22, 2018,33,250 bytes, machine format IBM-PC, MS-Windows operating system, ishereby incorporated by reference.

BACKGROUND

Complete remedy of HIV infection is often impeded by the presence oflatent virus in some cells. The latent virus may stay episomal orintegrated into the host cell's genome, remaining transcriptionallyinactive provirus. These infected cells that have latent virus canpersist for years without producing viral progeny, rendering themrefractory to immune surveillance and antiretroviral therapy andproviding a reservoir for reactivation and re propagation of HIV.Therefore there is a need to selectively remove such infected cells soas to purge the latent reservoir and completely eradicate infection.

BRIEF SUMMARY

In one aspect, provided herein is a recombinant peptide having a zincfinger domain. The recombinant peptide binds to a target nucleotidesequence and the target nucleotide sequence has the sequence of SEQ IDNO. 1 or a derivative thereof having at least 75% nucleotide sequenceidentity to the sequence of SEQ ID NO. 1.

In another aspect, provided herein is a nucleotide sequence encoding anyof the recombinant peptides of the present disclosure.

In another aspect, provided herein is an expression vector having theforegoing nucleotide sequence that encodes any of the recombinantpeptides of the present disclosure.

In another aspect, provided herein is a method of activating a latentHIV from a cell. The method has administering any of the recombinantpeptides or any of the expression vectors of the present disclosure tothe cell.

In another aspect, provided herein is a method of treating HumanImmunodeficiency Virus (HIV) in a subject in need thereof. The methodhas administering any of the recombinant peptides or any of theexpression vectors of the present disclosure to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D: ZFP-362 protein conjugate directed transcriptionalactivation of latent HIV. (FIG. 1A) Three iterations of the ZFP-362recombinant protein were developed. The top candidate ZFP-362, targetedto the LTR-362 site (SEQ ID NO:1) was experimentally tested andredesigned and developed with the TAT peptide, various NLSs and themaltose binding domain (MBP), e.g. made to function as a solubleprotein. (FIG. 1B) The ZFP-362-VPR was contrasted with dCas-VPR, foundpreviously to activate latent HIV at levels greater than and morespecific than the standard of care latency reactivating drugs. (FIG. 1C)ZFP-362-VPR activates LTR expression of GFP in PMO cells but (FIG. 1D)has no effect on PMO cells containing a deletion in the LTR-362 site(SEQ ID NO:1) targeted by ZFP-362-VPR. For FIGS. 1B-1D, the averages oftriplicate treated cells are shown with standard deviations and (*)which represents p<0.001 from a paired T-test.

FIG. 2 : Vector map of ZFP-362-VPR. The ZFP-362-VPR recombinant proteincan be expressed from the CMV promoter and contains a maltose bindingprotein (MBP) with a factor Xa cleavage site for purification ofrecombinant protein, the Tat peptide (Tat protein) for nuclear targetingand transit of the recombinant protein through the blood brain barrier,three nucleoplasmin nuclear localization signals (NLS) for enhancednuclear targeting of the recombinant protein, the ZFP-362 targeted tothe NF-kB doublet in the HIV LTR and the VPR transcriptional activatordomain (VP64+RelA (p65) and Rta AD). The entire protein is terminated bythe bGH poly A signal.

FIG. 3 : CHIP analysis of ZFP-362-VPR and dCas-VPR binding to the HIVLTR. HEK PMO cells (containing an integrated HIV vector which expressesGFP) were transfected with either small guide RNA (sg) and dCas-VPR orZFP-362-VPR and CHIP performed 72 hrs post-transfection to determinebinding to the LTR-362 site. The average to triplicate treated cells areshown with the standard deviations and P values from a paired T-test.

FIG. 4 : Effects of Recombinant ZFP-362-VPR expression on J-Lat latentinfected cell models. Three different J-Lat cell lines were subjected toeither transfection of gRNA F2/dCas-VPR or ZFP-362-VPR and controls orexposed directly to recombinant purified ZFP-362-VPR and controls. Theexpression of GFP was determined by FACS 72 hrs post-treatment. Theresults of triplicate treated cultures are shown with the standarddeviations. Statistically significant differences, as determined from apaired T-test are also shown (*) P<0.05 and (**) P<0.005.

FIGS. 5A-5B: Off-target loci for ZFP-362-VPR vs. gRNA F2-dCas-VPR. (FIG.5A) Venn diagram of distinct and overlapping off-target loci bound byZFP-362-VPR and gRNA F2-dCas-VPR. (FIG. 5B) Clustering of theZFP-362-VPR and gRNA F2-dCas-VPR and control pcDNA treated cells. ForFIGS. 5A-5B, triplicate treated cells were treated and 72 hrs later CHiPperformed with Anti-Myc tag antibody Abcam (ab9132) followed by Highthroughput deep sequencing.

FIG. 6 shows Table 1, illustrating top ZFP-362-VPR off-target genepromoter bound sites.

FIG. 7 shows Table 2, illustrating top F2-gRNA-362-dCasVPR off-targetgene promoter bound sites.

FIG. 8 is a bar graph illustrating activation of clad-specific LTRs inHIV-1 subtypes A, B, C, D, E, F, and G. For subtypes B-E, the averagesof triplicate treated cells are shown with standard deviations. In eachpair of bars, the first bar of the pair corresponds to results for a ZFPcontrol, and the right bar of the pair corresponds to results forZFP-362-VPR.

FIG. 9 illustrates alignments between various HIV subtypes relative tosubtype B at the LTR-362 site (SEQ ID NO:1) targeted by ZFP-362. Panel(A) shows an alignment of sequences from subtypes A, B, D, E, F, and G.Panel (B) shows an alignment of sequences from subtypes B and C, andillustrates two possible binding sites for ZFP-362. The accession numberfor each subtype sequence is indicated.

DETAILED DESCRIPTION

Disclosed herein are, inter alia, compositions and methods foractiviating latent Human Immunodeciency Virus (HIV) in cells. In oneaspect, the compositions and methods utilize a recombinant peptide thathas a DNA-binding, zinc finger domain and a transcription factor (e.g. atranscription activator) that is conjuated to the zinc finger domain.With this configuration, the specific recognition and binding of thezinc finger domain to its target nucleic acid sequence such as asequence from HIV genome results in recruiting the transcriptionactivator to the HIV genome, activating (e.g. initiating or increasing)the transcription of viral sequences. Also disclosed herein are, interalia, methods of treating HIV in a subject in need of the treatment. Themethod may involve activation of latent HIV in cells of the subject andselectively removing such cells from the subject, providing complete andeffective treatment of HIV.

Definitions

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by a person of ordinaryskill in the art. See, e.g., Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY 2nd ed., J. Wiley & Sons (New York,N.Y. 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL,Cold Springs Harbor Press (Cold Springs Harbor, N.Y. 1989). Any methods,devices and materials similar or equivalent to those described hereincan be used in the practice of this invention. The following definitionsare provided to facilitate understanding of certain terms usedfrequently herein and are not meant to limit the scope of the presentdisclosure.

As may be used herein, the terms “nucleic acid,” “nucleic acidmolecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acidsequence,” “nucleic acid fragment,” and “polynucleotide” are usedinterchangeably and are intended to include, but are not limited to, apolymeric form of nucleotides covalently linked together that may havevarious lengths, either deoxyribonucleotides or ribonucleotides, oranalogs, derivatives or modifications thereof.

Different polynucleotides may have different three-dimensionalstructures and may perform various functions, known or unknown.Non-limiting examples of polynucleotides include a gene, a genefragment, an exon, an intron, intergenic DNA (including, withoutlimitation, heterochromatic DNA), messenger RNA (mRNA), transfer RNA,ribosomal RNA, a ribozyme, cDNA, a recombinant polynucleotide, abranched polynucleotide, a plasmid, a vector, isolated DNA of asequence, isolated RNA of a sequence, a nucleic acid probe, and aprimer. Polynucleotides useful in the methods of the disclosure may havenatural nucleic acid sequences and variants thereof, artificial nucleicacid sequences, or a combination of such sequences.

A polynucleotide is typically composed of a specific sequence of fournucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine(T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus,the term “polynucleotide sequence” is the alphabetical representation ofa polynucleotide molecule; alternatively, the term may be applied to thepolynucleotide molecule itself. This alphabetical representation can beinput into databases in a computer having a central processing unit andused for bioinformatics applications such as functional genomics andhomology searching. Polynucleotides may optionally include one or morenon-standard nucleotide(s), nucleotide analog(s) and/or modifiednucleotides.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. The terms“non-naturally occurring amino acid” and “unnatural amino acid” refer toamino acid analogs, synthetic amino acids, and amino acid mimetics whichare not found in nature.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

The terms “amino acid sequence,” “polypeptide,” “peptide,” and “protein”are used interchangeably herein to refer to a polymer of amino acidresidues. The terms apply to amino acid polymers in which one or moreamino acid residue is an artificial chemical mimetic of a correspondingnaturally occurring amino acid, as well as to naturally occurring aminoacid polymers and non-naturally occurring amino acid polymers. A “fusionprotein” refers to a chimeric protein encoding two or more separateprotein sequences that are recombinantly expressed as a single moiety.

An amino acid or nucleotide base “position” is denoted by a number thatsequentially identifies each amino acid (or nucleotide base) in thereference sequence based on its position relative to the N-terminus (or5′-end). Due to deletions, insertions, truncations, fusions, and thelike that must be taken into account when determining an optimalalignment, in general the amino acid residue number in a test sequencedetermined by simply counting from the N-terminus will not necessarilybe the same as the number of its corresponding position in the referencesequence. For example, in a case where a variant has a deletion relativeto an aligned reference sequence, there will be no amino acid in thevariant that corresponds to a position in the reference sequence at thesite of deletion. Where there is an insertion in an aligned referencesequence, that insertion will not correspond to a numbered amino acidposition in the reference sequence. In the case of truncations orfusions there can be stretches of amino acids in either the reference oraligned sequence that do not correspond to any amino acid in thecorresponding sequence.

The terms “numbered with reference to” or “corresponding to,” when usedin the context of the numbering of a given amino acid or polynucleotidesequence, refers to the numbering of the residues of a specifiedreference sequence when the given amino acid or polynucleotide sequenceis compared to the reference sequence.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, “conservatively modified variants” refers to those nucleicacids that encode identical or essentially identical amino acidsequences. Because of the degeneracy of the genetic code, a number ofnucleic acid sequences will encode any given protein. For instance, thecodons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, atevery position where an alanine is specified by a codon, the codon canbe altered to any of the corresponding codons described without alteringthe encoded polypeptide. Such nucleic acid variations are “silentvariations,” which are one species of conservatively modifiedvariations. Every nucleic acid sequence herein which encodes apolypeptide also describes every possible silent variation of thenucleic acid. One of skill will recognize that each codon in a nucleicacid (except AUG, which is ordinarily the only codon for methionine, andTGG, which is ordinarily the only codon for tryptophan) can be modifiedto yield a functionally identical molecule. Accordingly, each silentvariation of a nucleic acid which encodes a polypeptide is implicit ineach described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the disclosure.

The following eight groups each contain amino acids that areconservative substitutions for one another:

-   1) Alanine (A), Glycine (G);-   2) Aspartic acid (D), Glutamic acid (E);-   3) Asparagine (N), Glutamine (Q);-   4) Arginine (R), Lysine (K);-   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);-   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);-   7) Serine (S), Threonine (T); and-   8) Cysteine (C), Methionine (M)

“Percentage of sequence identity” is determined by comparing twooptimally aligned sequences over a comparison window, wherein theportion of the polynucleotide or polypeptide sequence in the comparisonwindow may have additions or deletions (i.e., gaps) as compared to thereference sequence (which does not have additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

The terms “identical,” “percent identity,” “sequence identity,”“homology,” or “sequence homology” in the context of two or more nucleicacids or polypeptide sequences, refer to two or more sequences orsubsequences that are the same or have a specified percentage of aminoacid residues or nucleotides that are the same (i.e., about 60%identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or higher identity over a specified region,when compared and aligned for maximum correspondence over a comparisonwindow or designated region) as measured using a BLAST or BLAST 2.0sequence comparison algorithms with default parameters described below,or by manual alignment and visual inspection (see, e.g., NCBI web sitewww.ncbi.nlm.nih.gov/BLAST or the like). Such sequences are then said tobe “substantially identical.” This definition also refers to, or may beapplied to, the compliment of a test sequence. The definition alsoincludes sequences that have deletions and/or additions, as well asthose that have substitutions. As described below, the preferredalgorithms can account for gaps and the like. Preferably, identityexists over a region that is at least about 25 amino acids ornucleotides in length, or more preferably over a region that is 50-100amino acids or nucleotides in length. Optionally, the identity existsover a region that is at least about 50 nucleotides in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotidesin length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of, e.g., a full length sequence or from 20 to 600, about 50to about 200, or about 100 to about 150 amino acids or nucleotides inwhich a sequence may be compared to a reference sequence of the samenumber of contiguous positions after the two sequences are optimallyaligned. Methods of alignment of sequences for comparison are well knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith and Waterman(1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci.USA 85:2444, by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manualalignment and visual inspection (see, e.g., Ausubel et al., CurrentProtocols in Molecular Biology (1995 supplement)).

An example of an algorithm that is suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information(www.ncbi.nlm.nih.gov). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) or 10, M=5, N=−4 and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross-reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid. Thus,a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two molecules or theircomplements hybridize to each other under stringent conditions. Yetanother indication that two nucleic acid sequences are substantiallyidentical is that the same primers can be used to amplify the sequence.

The terms “variant” or “derivative” in the context of polynucleotide(e.g. nucleic acid sequence or oligonucleotide) or peptide (e.g. anamino acid sequence or protein) may refer to a polynucleotide sequenceor peptide sequence that has some sequence similarity to their referencesequence. In some examples, a variant or derivative can have at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% sequence identity (or equivalently used withsimilarity or homology) to its reference sequence. The terms “functionalderivative” or “functional variant” in the context of polynucleotide orpeptide sequence may refer to any variant or derivative that maintainsthe activity to a substantial level, e.g. at least 30% or more of theactivity of the reference sequence.

A “vector” as used herein is a nucleic acid molecule that can be used asa vehicle to transfer genetic material into a cell. In embodiments, avector refers to a DNA molecule harboring at least one origin ofreplication, a multiple cloning site (MCS) and one or more selectionmarkers. A vector is typically composed of a backbone region and atleast one insert or transgene region or a region designed for insertionof a DNA fragment or transgene such as a MCS. The backbone region oftencontains an origin of replication for propagation in at least one hostand one or more selection markers. A vector can have one or morerestriction endonuclease recognition sites (e.g., two, three, four,five, seven, ten, etc.) at which the sequences can be cut in adeterminable fashion without loss of an essential biological function ofthe vector, and into which a nucleic acid fragment can be spliced inorder to bring about its replication and cloning. Vectors can furtherprovide primer sites (e.g., for PCR), transcriptional and/ortranslational initiation and/or regulation sites, recombinationalsignals, replicons, selectable markers, etc. Clearly, methods ofinserting a desired nucleic acid fragment which do not require the useof recombination, transpositions or restriction enzymes (such as, butnot limited to, uracil N glycosylase (UDG) cloning of PCR fragments(U.S. Pat. Nos. 5,334,575 and 5,888,795, both of which are entirelyincorporated herein by reference), T:A cloning, and the like) can alsobe applied to clone a fragment into a cloning vector to be usedaccording to the present invention. In embodiments, a vector containsadditional features. Such additional features may include natural orsynthetic promoters, genetic markers, antibiotic resistance cassettes orselection markers (e.g., toxins such as ccdB or tse2), epitopes or tagsfor detection, manipulation or purification (e.g., V5 epitope, c-myc,hemagglutinin (HA), FLAG™, polyhistidine (His),glutathione-S-transferase (GST), maltose binding protein (MBP)),scaffold attachment regions (SARs) or reporter genes (e.g., greenfluorescent protein (GFP), red fluorescence protein (RFP), luciferase,β-galactosidase etc.). In embodiments, vectors are used to isolate,multiply or express inserted DNA fragments in a target host.

An “expression vector” is designed for expression of a transgene andgenerally harbors at least one promoter sequence that drives expressionof the transgene. Expression as used herein refers to transcription of atransgene or transcription and translation of an open reading frame andcan occur in a cell-free environment such as a cell-free expressionsystem or in a host cell. In embodiments expression of an open readingframe or a gene results in the production of a polypeptide or protein.An expression vector is typically designed to contain one or moreregulatory sequences such as enhancer, promoter and terminator regionsthat control expression of the inserted transgene. Suitable expressionvectors include, without limitation, plasmids and viral vectors. Vectorsand expression systems for various applications are available fromcommercial suppliers such as Novagen (Madison, Wis.), Clontech (PaloAlto, Calif.), Stratagene (La Jolla, Calif.), and Life TechnologiesCorp. (Carlsbad, Calif.).

A “promoter” as used herein is a transcription regulatory sequence whichis capable of directing transcription of a nucleic acid segment (e.g., atransgene having, for example, an open reading frame) when operablyconnected thereto. The choice of a promoter to be included in anexpression vector depends upon several factors, including withoutlimitation efficiency, selectability, inducibility, desired expressionlevel, and cell or tissue specificity. For example, tissue-, organ- andcell-specific promoters that confer transcription only or predominantlyin a particular tissue, organ, and cell type, respectively, can be used.Other classes of promoters include, but are not limited to, induciblepromoters, such as promoters that confer transcription in response toexternal stimuli such as chemical agents, developmental stimuli, orenvironmental stimuli. Inducible promoters may be induced by pathogensor stress like cold, heat, UV light, or high ionic concentrations or maybe induced by chemicals. Examples of inducible promoters are theeukaryotic metallothionein promoter, which is induced by increasedlevels of heavy metals; the prokaryotic lacL promoter, which is inducedin response to isopropyl-β-D-thiogalacto-pyranoside (IPTG); andeukaryotic heat shock promoters, which are induced by raisedtemperature. Numerous additional bacterial and eukaryotic promoterssuitable for use with the invention are known in the art and describedin, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual(2nd ed. 1989; 3rd ed., 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Ausubel et al., Current Protocols inMolecular Biology. Bacterial expression systems for expressing the ZFPare available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva etal. Secretion of interferon by Bacillus subtilis. Gene 22:229-235(1983)). Kits for such expression systems are commercially available.Eukaryotic expression systems for mammalian cells, yeast, and insectcells are well known by those of skill in the art and are alsocommercially available.

Common promoters for prokaryotic protein expression are e.g., lacpromoter or trc and tac promoter (IPTG induction), tetApromoter/operator (anhydrotetracyclin induction), PPBAD promoter(L-arabinose induction), r/zaPBAD promoter (L-rhamnose induction) orphage promoters such as phage promoter pL (temperature shift sensitive),T7, T3, SP6, or T5.

Common promoters for mammalian protein expression are, e.g.,Cytomegalovirus (CMV) promoter, H1 promoter, EF1 alpha promoter, SV40promoter/enhancer, Vaccinia virus promoter, Viral LTRs (MMTV, RSV, HIVetc.), E1B promoter, promoters of constitutively expressed genes (actin,GAPDH), promoters of genes expressed in a tissue-specific manner(albumin, NSE), promoters of inducible genes (Metallothionein, steroidhormones).

The term “isolated,” when applied to a nucleic acid or protein, denotesthat the nucleic acid or protein is essentially free of other cellularcomponents with which it is associated in the natural state. It can be,for example, in a homogeneous state and may be in either a dry oraqueous solution. Purity and homogeneity are typically determined usinganalytical chemistry techniques such as polyacrylamide gelelectrophoresis or high performance liquid chromatography. A proteinthat is the predominant species present in a preparation issubstantially purified.

The term “purified” denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. In embodiments, thenucleic acid or protein is at least 50% pure, optionally at least 65%pure, optionally at least 75% pure, optionally at least 85% pure,optionally at least 95% pure, and optionally at least 99% pure.

As used herein, the phrase “recombination proteins” includes excisive orintegrative proteins, enzymes, co-factors or associated proteins thatare involved in recombination reactions involving one or morerecombination sites (e.g., two, three, four, five, seven, ten, twelve,fifteen, twenty, thirty, fifty, etc.), which may be wild-type proteins(see Landy, Current Opinion in Biotechnology 3:699-707 (1993)), ormutants, derivatives (e.g., fusion proteins containing the recombinationprotein sequences or fragments thereof), fragments, and variantsthereof. Examples of recombination proteins include Cre, Int, IHF, Xis,Flp, Fis, Hin, Gin, Phi-C31, CM, Tn3 resolvase, TndX, XerC, XerD, TnpX,Hjc, SpCCEl, and Par A.

As used herein, the term “conjugate” refers to the association betweenatoms or molecules. The association can be direct or indirect. Forexample, a conjugate between a first moiety (e.g., DNA binding domain)and a second moiety (e.g. a transcriptional modulator such astranscription activator) provided herein can be direct, e.g., bycovalent bond, or indirect, e.g., by non-covalent bond (e.g.electrostatic interactions (e.g. ionic bond, hydrogen bond, halogenbond), van der Waals interactions (e.g. dipole-dipole, dipole-induceddipole, London dispersion), ring stacking (pi effects), hydrophobicinteractions and the like). In embodiments, conjugates are formed usingconjugate chemistry including, but are not limited to nucleophilicsubstitutions (e.g., reactions of amines and alcohols with acyl halides,active esters), electrophilic substitutions (e.g., enamine reactions)and additions to carbon-carbon and carbon-heteroatom multiple bonds(e.g., Michael reaction, Diels-Alder addition). In embodiments, a firstmoiety and a second moiety in a conjugate are linked via a peptidelinker, e.g. a polymer of amino acids having about 1 to 300 amino acids.Some examples of such a linker include one or more repeat of glycine andserine, e.g. (Gly-Gly-Gly-Ser)n, wherein n is 1 or higher integer. Anylinkers known in the art (e.g. see parts.igem.org/Proteindomains/Linker) can be used in the recombinant peptides and conjugatesof the disclosure. Alternatively, first and second moieties in aconjugate are linked directly without any linking amino acids. These andother useful reactions are discussed in, for example, March, ADVANCEDORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985;Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; andFeeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series,Vol. 198, American Chemical Society, Washington, D.C., 1982.

A “cell” as used herein, refers to a cell carrying out metabolic orother function sufficient to preserve or replicate its genomic DNA. Acell can be identified by well-known methods in the art including, forexample, presence of an intact membrane, staining by a particular dye,ability to produce progeny or, in the case of a gamete, ability tocombine with a second gamete to produce a viable offspring. Cells mayinclude prokaryotic and eukaryotic cells. Prokaryotic cells include butare not limited to bacteria. Eukaryotic cells include but are notlimited to yeast cells and cells derived from plants and animals, forexample mammalian, insect (e.g., spodoptera) and human cells.

The term “expression” or “expressed” as used herein in reference to agene means the transcriptional and/or translational product of thatgene. The level of expression of a DNA molecule in a cell may bedetermined on the basis of either the amount of corresponding mRNA thatis present within the cell or the amount of protein encoded by that DNAproduced by the cell (Sambrook et al., 1989, Molecular Cloning: ALaboratory Manual, 18.1-18.88).

Expression of a transfected gene can occur transiently or stably in acell. During “transient expression” the transfected gene is nottransferred to the daughter cell during cell division. Since itsexpression is restricted to the transfected cell, expression of the geneis lost over time. In contrast, stable expression of a transfected genecan occur when the gene is co-transfected with another gene that confersa selection advantage to the transfected cell. Such a selectionadvantage may be a resistance towards a certain toxin that is presentedto the cell.

The terms “transfection,” “transduction,” “transfecting,” or“transducing” can be used interchangeably and are defined as a processof introducing a nucleic acid molecule and/or a protein to a cell.Nucleic acids may be introduced to a cell using various methods. Thenucleic acid molecule can be a sequence encoding complete proteins orfunctional portions thereof. Typically, a nucleic acid vector,comprising the elements necessary for protein expression (e.g., apromoter, transcription start site, etc.). Exemplary transfectionmethods include calcium phosphate transfection, liposomal transfection,nucleofection, sonoporation, transfection through heat shock,magnetifection and electroporation. The terms “transfection” or“transduction” also refer to introducing proteins into a cell from theexternal environment. Typically, transduction or transfection of aprotein relies on attachment of a peptide or protein capable of crossingthe cell membrane to the protein of interest. See, e.g., Ford et al.(2001) Gene Therapy 8:1-4 and Prochiantz (2007) Nat. Methods 4:119-20.

As used herein, the terms “specific binding,” “specifically bind,” or“specifically binds” refer to two molecules (e.g., DNA-binding domainand its specific binding (or targeting) nucleic acid sequence) that bindto each other with a higher affinity and specificity than a bindingbetween random (e.g. non-target) molecules.

As used herein, the phrase “recognition sequence,” “recognition site,”“target sequence” or “target site” refers to a particular sequence towhich a protein, chemical compound, DNA, or RNA molecule (e.g., a DNAbinding domain such as zinc finger domain) recognizes and binds. Arecognition sequence or target sequence may refer to a nucleic acidsequence, DNA or RNA, that is recognized and bound by a recombinantpeptide with specificity. In certain examples, the recognition sequenceor target sequence may be a nucleic acid sequence from HIV genome thatis either integrated into the host cell's genome or present as aseparate nucleic acid molecule (i.e. episome). Therefore, in someexamples where the recombinant peptide has transcription activity, thetranscription of the recognition sequence (or the target sequence) or asequence having the recognition sequence (or the target sequence) can beactivated by the recombinant peptide.

As defined herein, the term “activation,” “activate,” “activating” andthe like in reference to gene expression or transcription refers toconversion of a gene or nucleic acid sequence to be transcribed to itscomplementary RNA (e.g. mRNA) from an initially inactive or deactivatedstate. In some cases, the gene or nucleic acid sequence that is normallytranscribed to a certain extent is activated such that its transcriptionlevel is enhanced or increased as compared to its normal level oftranscription.

The term “activation,” “activate,” “activating,” “reactivation,”“reactivate,” “reactivating” and the like used in the context of virusinfection refers to enhancing, promoting, stimulating or increasing theactivity of the virus including the transcriptional activation of viralgene(s) or genome. Especially in the context of activation orreactivation of latent virus, the term may refer to (i) initiation oftranscription of certain viral genes that were previouslytranscriptionally inactive, (ii) increase of existing transcription ofviral genes and/or (iii) transcription of a viral genome. With thisactivation or reactivation, the latent virus may becometranscriptionally active, replicating the viral genome and producingviral progenies such that the virus is no longer in the latency andenters into the lytic cycle.

“Treating” and “treatment” as used herein include administering to asubject a therapeutically effective amount of an active agent. Theadministering step may consist of a single administration or may includea series of administrations. The length of the treatment period dependson a variety of factors, such as the severity of the condition, the ageof the patient, the concentration of active agent, the activity of thecompositions used in the treatment, or a combination thereof. It willalso be appreciated that the effective dosage of an agent used for thetreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent by standarddiagnostic assays known in the art. In some instances, chronicadministration may be required. For example, the compositions areadministered to the subject in an amount and for a duration sufficientto treat the patient.

“Patient” or “subject in need thereof” refers to a living organismsuffering from or prone to a disease or condition that can be treated byadministration of a pharmaceutical composition as provided herein.Non-limiting examples include humans, other mammals, bovines, rats,mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammaliananimals. In embodiments, a patient is human.

A “effective amount” is an amount sufficient for a compound toaccomplish a stated purpose relative to the absence of the compound(e.g. achieve the effect for which it is administered, treat a diseaseor reduce one or more symptoms of a disease or condition). An example ofan “effective amount” is an amount sufficient to contribute to thetreatment, prevention, or reduction of a symptom or symptoms of adisease, which could also be referred to as a “therapeutically effectiveamount.” The exact amounts will depend on the purpose of the treatment,and will be ascertainable by one skilled in the art using knowntechniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of PharmaceuticalCompounding (1999); Pickar, Dosage Calculations (1999); and Remington:The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed.,Lippincott, Williams & Wilkins).

Dosages may be varied depending upon the requirements of the patient andthe compound being employed. The dose administered to a patient, in thecontext of the present disclosure, should be sufficient to affect abeneficial therapeutic response in the patient over time. The size ofthe dose also will be determined by the existence, nature, and extent ofany adverse side-effects. Determination of the proper dosage for aparticular situation is within the skill of the practitioner. Generally,treatment is initiated with smaller dosages which are less than theoptimum dose of the compound. Thereafter, the dosage is increased bysmall increments until the optimum effect under circumstances isreached. Dosage amounts and intervals can be adjusted individually toprovide levels of the administered compound effective for the particularclinical indication being treated. This will provide a therapeuticregimen that is commensurate with the severity of the individual'sdisease state.

As used herein, the term “administering” means oral administration,administration as a suppository, topical contact, intravenous,parenteral, intraperitoneal, intramuscular, intralesional, intrathecal,intranasal or subcutaneous administration, or the implantation of aslow-release device, e.g., a mini-osmotic pump, to a subject.Administration is by any route, including parenteral and transmucosal(e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, ortransdermal). Parenteral administration includes, e.g., intravenous,intramuscular, intra-arteriole, intradermal, subcutaneous,intraperitoneal, intraventricular, and intracranial. Other modes ofdelivery include, but are not limited to, the use of liposomalformulations, intravenous infusion, transdermal patches, etc. Inembodiments, the administering does not include administration of anyactive agent other than the recited active agent.

“Co-administer” it is meant that a composition described herein isadministered at the same time, just prior to, or just after theadministration of one or more additional therapies. The compoundsprovided herein can be administered alone or can be coadministered tothe patient. Coadministration is meant to include simultaneous orsequential administration of the compounds individually or incombination (more than one compound). Thus, the preparations can also becombined, when desired, with other active substances (e.g. to reducemetabolic degradation). The compositions of the present disclosure canbe delivered transdermally, by a topical route, or formulated asapplicator sticks, solutions, suspensions, emulsions, gels, creams,ointments, pastes, jellies, paints, powders, and aerosols.

As used herein, the term “about” means a range of values including thespecified value, which a person of ordinary skill in the art wouldconsider reasonably similar to the specified value. In embodiments, theterm “about” means within a standard deviation using measurementsgenerally acceptable in the art. In embodiments, about means a rangeextending to +/−10% of the specified value. In embodiments, about meansthe specified value.

Throughout this document, unless the context requires otherwise, thewords “comprise,” “comprising,” “contain,” “containing,” “have,”“having,” “include,” or “including” will be understood to imply theinclusion of a stated step or element or group of steps or elements butnot the exclusion of any other step or element or group of steps orelements.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

Certain virus has two cycles of reproduction which are the lytic cycleand lysogenic cycles. The lytic cycle results in the destruction of theinfected cell and its membrane. One of major differences between thelytic and lysogenic phage cycles is that in the lytic phage, the viralDNA may exist as a separate molecule within the bacterial cell, andreplicates separately from the host bacterial DNA. The location of viralDNA in the lysogenic phage cycle may be within the host DNA, thereforein both cases the virus/phage replicates using the host DNA machinery,but in the lytic phage cycle, the phage is a free floating separatemolecule to the host DNA. However, in some cases, even during thelysogenic cycle, the viral genome is not integrated into the host DNAbut exists as a separate molecule as an episome. When the virus is inlysogeny or lysogenic cycle, the host cell can continue to live andreproduce normally. The genetic material of the virus in the lysogeniccycle, i.e. latent virus, can be transmitted to daughter cells at eachsubsequent cell division, and at later events (such as UV radiation orthe presence of certain chemicals) can release it, causing proliferationof new phages via the lytic cycle.

Virus latency (or viral latency) is the ability of a pathogenic virus tolie dormant (latent) within a cell, denoted as the lysogenic part of theviral life cycle. Latency is often the phase in certain viruses' lifecycles in which, after initial infection, proliferation of virusparticles ceases. However, the viral genome is not fully eradicated. Theresult of this is that the virus can reactivate and begin producinglarge amounts of viral progeny without the host being infected by newoutside virus, denoted as the lytic part of the viral life cycle, andstays within the host for a long period or even indefinitely.

Depending on the location of the viral genome in the host cell duringthe latency, there are generally two types of latency, episomal latencyand proviral latency. Episomal latency refers to the use of geneticepisomes during latency. In this type, viral genes are stabilizedfloating in the cytoplasm or nucleus as distinct objects, both as linearor lariat structures. Alternatively, the latent virus can be a provirusthat is a virus genome which is integrated into the DNA of a host cell.

One of the well-known virus for the latent capability is HIV. In theproviral latency, HIV uses reverse transcriptase to create a DNA copy ofits RNA genome. This allows the virus to largely avoid the immunesystem. Like other viruses that go latent, it does not typically causesymptoms while latent. Unfortunately, HIV in proviral latency is nearlyimpossible to target with existing antiretroviral drugs. Given thelatent HIV can be active and pathogenic, i.e. entering into lytic cyclelater times, the patient with the latent virus in his or her systemstill has a risk of suffering from pathogenic, reactivated HIV infectionat later times. Therefore, removing such infected cells from thepatient's system is important in order to completely and effectivelytreat HIV in the patient.

In embodiments, provided is an engineered transcriptional activationsystem and method based on a recombinant zinc finger peptide which canselectively activate HIV viral gene transcription and expression incells of HIV latency. In embodiments, the system and method utilize arecombinant peptide having a zinc finger domain and a transcriptionactivator. In embodiments, the zinc finger domain targets (or binds to)a sequence of HIV genome. This specific recognition of, and binding to,the HIV sequence by the zinc finger domain results in recruiting theconjugated transcription activator to the latent HIV genome, activatingthe transcription of HIV gene(s) and reactivating the latent HIV. Inembodiments, the system and method are effective tools for inducinglatent HIV transcription and expression and that their use. Inembodiments, the system and method may be used in combination withantiretroviral therapy to provide improved therapies for HIV infection.In embodiments, the HIV is an HIV subtype of A, B, C, D, E, F, or G. Inembodiments, the HIV subtype is A or B. A “subtype” may also be referredto as a “clade.”

Compositions

In one aspect, the present disclosure provides a recombinant peptidethat has a zinc finger domain. A zinc finger domain is used hereinaccording to its plain and ordinary meaning in the art and generallyrefers to a protein structural domain capable of binding a targetnucleic acid sequence. In embodiments, the zinc finger domaincoordinates one or more zinc ions in order to stabilize its structure.Zinc finger (Znf) domains may be relatively small protein domains thatcan contain multiple finger-like protrusions that make tandem contactswith their target molecule. In embodiments, the Znf forms salt bridgesto stabilize the finger-like folds. In embodiments, the Znf formscoordinates a metal (e.g. zinc) to stabilize the finger-like folds.

In embodiments, the zinc finger domain that is used in the recombinantpeptide of the disclosures recognizes (or binds to) a target nucleicacid sequence with specificity. The target nucleic acid can be DNA orRNA sequence. In embodiments, the target nucleic acid sequence containsthe sequence of SEQ ID NO. 1 or a derivative of the sequence of SEQ IDNO. 1. In embodiments, the derivative is a nucleic acid sequence thathas at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98% or 99% sequence identity to the sequence of SEQ ID NO.1. Inembodiments, the derivative has at least 90%, 95%, 96%, 97%, 98%, 99% or100% nucleic acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 5, 10, 15 or 20 continuous nucleic acidportion) compared to SEQ ID NO. 1.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.1, whereinthe derivative has at least 85% nucleotide sequence identity to thesequence of SEQ ID NO. 1.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.1, whereinthe derivative has at least 90% nucleotide sequence identity to thesequence of SEQ ID NO. 1.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.1, whereinthe derivative has at least 95% nucleotide sequence identity to thesequence of SEQ ID NO. 1.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.1, whereinthe derivative has at least 96% nucleotide sequence identity to thesequence of SEQ ID NO. 1.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.1, whereinthe derivative has at least 97% nucleotide sequence identity to thesequence of SEQ ID NO. 1.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.1, whereinthe derivative has at least 98% nucleotide sequence identity to thesequence of SEQ ID NO. 1.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.1, whereinthe derivative has at least 99% nucleotide sequence identity to thesequence of SEQ ID NO. 1.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain contains the sequence of SEQ ID NO. 2 or a derivative of thesequence of SEQ ID NO. 2. The derivative herein may refer to a nucleicacid sequence that has the nucleotide sequence identity of at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% to thesequence of SEQ ID NO.2. In embodiments, the derivative has at least90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 10, 50,100, 150, 200 or more continuous nucleic acid portion) compared to SEQID NO. 2.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.2, whereinthe derivative has at least 85% nucleotide sequence identity to thesequence of SEQ ID NO. 2.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.2, whereinthe derivative has at least 90% nucleotide sequence identity to thesequence of SEQ ID NO. 2.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.2, whereinthe derivative has at least 95% nucleotide sequence identity to thesequence of SEQ ID NO. 2.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.2, whereinthe derivative has at least 96% nucleotide sequence identity to thesequence of SEQ ID NO. 2.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.2, whereinthe derivative has at least 97% nucleotide sequence identity to thesequence of SEQ ID NO. 2.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.2, whereinthe derivative has at least 98% nucleotide sequence identity to thesequence of SEQ ID NO. 2.

In embodiments, the target nucleic acid sequence of the zinc fingerdomain has a derivative sequence of the sequence of SEQ ID NO.2, whereinthe derivative has at least 99% nucleotide sequence identity to thesequence of SEQ ID NO. 2.

In embodiments, the zinc finger domain of the recombinant peptiderecognizes and binds to a sequence of HIV genome with specificity. Inembodiments, the HIV genome sequence that is specifically recognized (orbound) by the zinc finger domain is a long terminal repeat (LTR) of HIV.

Long terminal repeats (LTRs) are used according to their plain andordinary meaning and the art. Thus, LTR's may contain identicalsequences of DNA or RNA that repeat hundreds or thousands of times foundat either end of viral retroviral genome or proviral DNA that is formedby reverse transcription of retroviral RNA. They may be used by virusesto insert their genetic material into the host genomes. The LTRs may bepartially transcribed into an RNA intermediate, followed by reversetranscription into complementary DNA (cDNA) and ultimately dsDNA(double-stranded DNA) with full LTRs. The LTRs may then mediateintegration of the retroviral DNA via an LTR specific integrase intoanother region of the host chromosome. In the proviral latency, once theprovirus has been integrated, the LTR on the 5′ end may serve as thepromoter for the entire retroviral genome, while the LTR at the 3′ endmay provide for nascent viral RNA polyadenylation and encodes someaccessory proteins. In embodiments, the recombinant peptide of thepresent disclosure targets (or binds to) 5′ LTR, 3′ LTR or both.

In embodiments, the zinc finger domain recognizes with specificity (e.g.specifically binds) about 5 bases, about 10 bases, about 15 bases, about20 bases, about 25 bases, about 30 bases, about 35 bases, about 40bases, about 45 bases or about 50 bases from the HIV LTR sequence of arecognized sequence (e.g. the target sequence). In embodiments, the zincfinger domain recognizes (e.g. binds to) a derivative of the targetsequence which has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identify to the target sequence.

In embodiments, the recombinant peptide of the present disclosureincludes one or more than one zinc finger domains. In embodiments, thenumber of zinc finger domains present in a single molecule of therecombinant peptide is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

In embodiments, the recombinant peptide having one or more zinc fingerdomains used in the present disclosure is encoded by the sequence of SEQID NO. 3 or a derivative thereof. The sequence of SEQ ID NO. 3 encodes anon-naturally occurring peptide sequence, which may be referred toherein as “ZFP-362” or “ZFP-362 peptide.” The amino acid sequenceencoded by SEQ ID NO. 3 is shown in SEQ ID NO. 12. In embodiments, theZFP-362 peptide specifically targets (e.g. specifically binds, or iscapable of specifically binding) the HIV LTR including HIV-1 LTR andinduces activation of HIV transcription at levels comparable todefective CRISPR VPR conjugates (dCas-VPR) (see FIGS. 1B and 1C). Inembodiments, the targeted activation of HIV LTR expression by theZFP-362 conjugated to VPR (also referred to herein as ZFP-362-VPR),which is further described below, is specifically targeted to thewell-defined NF-κB double site, a region known to be susceptible tomodulation and control of viral transcription and only found in HIV. Inembodiments, targeting of ZFP-362-VPR to cells lacking to thewell-defined NF-κB double site are not activated (see FIG. 1D) comparedwith those containing this site (see FIG. 1C). Therefore, inembodiments, the recombinant peptide of the present disclosure that hasthe ZFP-362 or derivative thereof can induce transcriptional activationin the cells infected with latent HIV, but not in HIV-free cells. Inembodiments, the infected cells from which the latent HIV are activatedby the recombinant peptide of the disclosures will be treated, e.g.killed by the action of additional anti-viral drugs.

In embodiments, the recombinant peptide of the present disclosure isencoded by the sequence of SEQ ID NO. 3 or a derivative thereof. Inembodiments, the derivative has at least 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequenceidentity to the sequence of SEQ ID NO. 3. In embodiments, the derivativehas at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequenceidentity across the whole sequence or a portion of the sequence (e.g. a20, 50, 100, 150, 200 or more continuous nucleic acid portion) comparedto SEQ ID NO. 3. Also, in embodiments, the derivative encodes afunctional derivative of the recombinant peptide encoded by SEQ ID NO. 3that has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% of the activity of the peptideencoded by SEQ ID NO. 3 (e.g. the activity of the ZFP-362 that binds toits target nucleic acid sequence with specificity).

In embodiments, the derivative of SEQ ID NO. 3 has at least 75% sequenceidentity to at least 500 contiguous nucleotides of SEQ ID NO. 3. Inembodiments, the derivative has at least 80% sequence identity to atleast 400 contiguous nucleotides of SEQ ID NO. 3. In embodiments, thederivative has at least 90% sequence identity to at least 300 contiguousnucleotides of SEQ ID NO. 3. In embodiments, the derivative has at least95% sequence identity to at least 200 contiguous nucleotides of SEQ IDNO. 3. In embodiments, the derivative has 100% identity to at least 100contiguous nucleotides of SEQ ID NO. 3. In embodiments, the derivativehas one or more conservative modifications of SEQ ID NO. 3 or of thepolypeptide it encodes (e.g., 1, 2, 3, 4, 5, 10, 15, 25, 50, 100, 150,200, or more conservative modifications).

In embodiments, the recombinant peptide of the present disclosure hasthe sequence of SEQ ID NO. 12 or a derivative thereof. In embodiments,the derivative has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to thesequence of SEQ ID NO. 12. In embodiments, the derivative has at least90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity across the wholesequence or a portion of the sequence (e.g. a 20, 50, 100, 150, 200 ormore continuous amino acid portion) compared to SEQ ID NO. 12. Also, inembodiments, the derivative is a functional derivative of therecombinant peptide of SEQ ID NO. 12 that has at least 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% of the activity of the polypeptide of SEQ ID NO. 12 (e.g. theactivity of the ZFP-362 that binds to its target nucleic acid sequencewith specificity).

In embodiments, the derivative of SEQ ID NO. 12 has at least 75%sequence identity to at least 150 contiguous amino acids of SEQ ID NO.12. In embodiments, the derivative has at least 80% sequence identity toat least 125 contiguous amino acids of SEQ ID NO. 12. In embodiments,the derivative has at least 90% sequence identity to at least 100contiguous amino acids of SEQ ID NO. 12. In embodiments, the derivativehas at least 95% sequence identity to at least 75 contiguous amino acidsof SEQ ID NO. 12. In embodiments, the derivative has 100% identity to atleast 50 contiguous amino acids of SEQ ID NO. 12. In embodiments, thederivative has one or more conservative modifications of SEQ ID NO. 12(e.g., 1, 2, 3, 4, 5, 10, 15, 25, 50, or more conservativemodifications).

In embodiments, the recombinant peptide of the present disclosure isencoded by the sequence of SEQ ID NO. 3 or a derivative thereof havingat least 75% nucleic acid sequence identity to the sequence of SEQ IDNO. 3. In embodiments, the recombinant peptide has the sequence of SEQID NO. 12, or a derivative thereof having at least 75% amino acidsequence identity to the sequence of SEQ ID NO. 12.

In embodiments, the recombinant peptide of the present disclosure isencoded by the sequence of SEQ ID NO. 3 or a derivative thereof havingat least 80% nucleic acid sequence identity to the sequence of SEQ IDNO. 3. In embodiments, the recombinant peptide has the sequence of SEQID NO. 12, or a derivative thereof having at least 80% amino acidsequence identity to the sequence of SEQ ID NO. 12.

In embodiments, the recombinant peptide of the present disclosure isencoded by the sequence of SEQ ID NO. 3 or a derivative thereof havingat least 85% nucleic acid sequence identity to the sequence of SEQ IDNO. 3. In embodiments, the recombinant peptide has the sequence of SEQID NO. 12, or a derivative thereof having at least 85% amino acidsequence identity to the sequence of SEQ ID NO. 12.

In embodiments, the recombinant peptide of the present disclosure isencoded by the sequence of SEQ ID NO. 3 or a derivative thereof havingat least 90% nucleic acid sequence identity to the sequence of SEQ IDNO. 3. In embodiments, the recombinant peptide has the sequence of SEQID NO. 12, or a derivative thereof having at least 90% amino acidsequence identity to the sequence of SEQ ID NO. 12.

In embodiments, the recombinant peptide of the present disclosure isencoded by the sequence of SEQ ID NO. 3 or a derivative thereof havingat least 95% nucleic acid sequence identity to the sequence of SEQ IDNO. 3. In embodiments, the recombinant peptide has the sequence of SEQID NO. 12, or a derivative thereof having at least 95% amino acidsequence identity to the sequence of SEQ ID NO. 12.

In embodiments, the recombinant peptide of the present disclosure isencoded by the sequence of SEQ ID NO. 3 or a derivative thereof havingat least 96% nucleic acid sequence identity to the sequence of SEQ IDNO. 3. In embodiments, the recombinant peptide has the sequence of SEQID NO. 12, or a derivative thereof having at least 96% amino acidsequence identity to the sequence of SEQ ID NO. 12.

In embodiments, the recombinant peptide of the present disclosure isencoded by the sequence of SEQ ID NO. 3 or a derivative thereof havingat least 97% nucleic acid sequence identity to the sequence of SEQ IDNO. 3. In embodiments, the recombinant peptide has the sequence of SEQID NO. 12, or a derivative thereof having at least 97% amino acidsequence identity to the sequence of SEQ ID NO. 12.

In embodiments, the recombinant peptide of the present disclosure isencoded by the sequence of SEQ ID NO. 3 or a derivative thereof havingat least 98% nucleic acid sequence identity to the sequence of SEQ IDNO. 3. In embodiments, the recombinant peptide has the sequence of SEQID NO. 12, or a derivative thereof having at least 98% amino acidsequence identity to the sequence of SEQ ID NO. 12.

In embodiments, the recombinant peptide of the present disclosure isencoded by the sequence of SEQ ID NO. 3 or a derivative thereof havingat least 99% nucleic acid sequence identity to the sequence of SEQ IDNO. 3. In embodiments, the recombinant peptide has the sequence of SEQID NO. 12, or a derivative thereof having at least 99% amino acidsequence identity to the sequence of SEQ ID NO. 12.

In embodiments, the recombinant peptide has one or more additionalcomponents such as additional peptides. In embodiments, the zinc fingerdomain can form a conjugate with the one or more peptides to form therecombinant peptide of the disclosures. The conjugate can be formed, forexample, via a chemical linkage such as a covalent bond or anon-chemical linkage such as ionic binding. In embodiments, the zincfinger domain and the additional peptide can be covalently linked toeach other, i.e. forming a fusion protein with or without a sequencelinking the two.

In some embodiment, the additional peptide that form a conjugate withthe zinc finger domain is a peptide capable of transcriptionalactivation. In embodiments, the peptide capable of transcriptionalactivation is a transcriptional activator. A transcriptional activatoris a protein (transcription factor) that increases gene transcription ofa gene or set of genes. In some cases, transcriptional activators, whenrecruited to a DNA site, e.g. a promoter of a target sequence foractivation, make or enhance protein-protein interactions with thegeneral transcription machinery (e.g. RNA polymerase and generaltranscription factors), thereby facilitating the binding of the generaltranscription machinery to the promoter.

Any peptide that is capable of activating transcription (e.g. initiatingthe transcription of a transcriptionally silent sequence or increasingthe transcription of an already transcriptionally active sequence) canbe used in the recombinant peptide of the disclosures. In embodiments,the transcriptional activators used in the present disclosure include,but not limited to, viral protein P (VPR), p65 transactivating subunitof NF-kappa B, heat-shock factor 1 (HSF) activation domain, VP64(tetramer of VP16) activation domain, synergistic activation mediator(SAM) and any derivatives thereof.

VPR is used herein according to its plain and ordinary meaning in theart. In embodiments, the recombinant peptide of the present disclosureincludes a VPR peptide or derivative thereof that is capable oftranscriptional activation. In embodiments, the VPR is encoded by thesequence of SEQ ID NO. 4 or a derivative thereof. In embodiments, thederivative has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the nucleic acidsequence identity to the sequence of SEQ ID NO. 4. In embodiments, thederivative has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleicacid sequence identity across the whole sequence or a portion of thesequence (e.g. a 20, 50, 100, 150, 200 or more continuous nucleic acidportion) compared to SEQ ID NO. 4. Also, in embodiments, the derivativeencodes a functional derivative of VPR that has at least 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% of the transcriptional activity of the VPR encoded by SEQ ID NO. 4.

In embodiments, the derivative of SEQ ID NO. 4 has at least 75% sequenceidentity to at least 1,300 contiguous nucleotides of SEQ ID NO. 4. Inembodiments, the derivative has at least 80% sequence identity to atleast 1,000 contiguous nucleotides of SEQ ID NO. 4. In embodiments, thederivative has at least 90% sequence identity to at least 750 contiguousnucleotides of SEQ ID NO. 4. In embodiments, the derivative has at least95% sequence identity to at least 500 contiguous nucleotides of SEQ IDNO. 4. In embodiments, the derivative has 100% sequence identity to atleast 250 contiguous nucleotides of SEQ ID NO. 4. In embodiments, thederivative has one or more conservative modifications of SEQ ID NO. 4 orof the polypeptide it encodes (e.g., 1, 2, 3, 4, 5, 10, 15, 25, 50, 100,150, 200, 300, 500, or more conservative modifications).

In embodiments, the VPR peptide has the sequence of SEQ ID NO. 13 or aderivative thereof. In embodiments, the derivative has at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity to the sequence of SEQ ID NO. 13. Inembodiments, the derivative has at least 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity across the whole sequence or a portion of thesequence (e.g. a 20, 50, 100, 150, 200 or more continuous amino acidportion) compared to SEQ ID NO. 13. Also, in embodiments, the derivativeis a functional derivative of the VPR of SEQ ID NO. 13 that has at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% of the activity of the polypeptide of SEQ ID NO.13.

In embodiments, the derivative of SEQ ID NO. 13 has at least 75%sequence identity to at least 400 contiguous amino acids of SEQ ID NO.13. In embodiments, the derivative has at least 80% sequence identity toat least 300 contiguous amino acids of SEQ ID NO. 13. In embodiments,the derivative has at least 90% sequence identity to at least 250contiguous amino acids of SEQ ID NO. 13. In embodiments, the derivativehas at least 95% sequence identity to at least 200 contiguous aminoacids of SEQ ID NO. 13. In embodiments, the derivative has 100% identityto at least 100 contiguous amino acids of SEQ ID NO. 13. In embodiments,the derivative has one or more conservative modifications of SEQ ID NO.13 (e.g., 1, 2, 3, 4, 5, 10, 15, 25, 50, 100, or more conservativemodifications).

In embodiments, the ZFP-362 or derivative thereof is conjugated to atranscriptional activator, forming the recombinant peptide of thepresent disclosure. In embodiments, the ZFP-362 or a derivative thereofis conjugated to a VPR or derivative thereof. In these embodiments, theZFP-362 specifically binds to its target sequence, e.g. the HIV LRTsequence and this binding will bring the VPR (or derivative thereof) tothe LRT sequence, promoting the transcription of LTR and the rest of HIVgenome. In embodiments, this specific binding activates HIV latentlypresent in the infected cells. In embodiments, the infected cells aretargeted with antiviral drugs in a follow-on treatment.

In embodiments, the recombinant peptide of the present disclosure hasfurther components, which includes, but are not limited to, acell-penetrating peptide (e.g. a TAT peptide or a derivative thereof)and/or one or more nuclear localization signals. Additionally, a peptidethat can promote stabilization of the recombinant protein and/or enhancethe protein isolation (e.g. myc-tag sequence and a maltose bindingsequence) can also be contained in the recombinant peptide.

Cell-penetrating peptides (CPPs) generally are short peptides that canfacilitate cellular intake/uptake of various molecular equipment (e.g. arecombinant peptide). The cargo is associated with the CPPs eitherthrough chemical linkage via covalent bonds or through non-covalentinteractions. The function of the CPPs is to deliver the cargo intocells. Any peptides that are known to be capable of CPPs or havecell-penetrating activity can be used in the composition and methods ofthe disclosures. In embodiments, the trans-activating transcriptionalactivator (TAT), which was initially found as a HIV-1 gene and productthereof, or a derivative of TAT is used as a CPP, thereby enhancing theintake/uptake of the recombinant peptide into the cells. In addition toenhancing the transfer to the nucleus of the cell, the TAT peptide canalso facilitate crossing the blood brain barrier, which can furtherenhance the delivery of the recombinant protein to the cells. Inembodiments, the TAT peptide used in the disclosure is encoded by thesequence of SEQ ID NO. 5 or a derivative thereof. In embodiments, thederivative has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the nucleic acidsequence identity to the sequence of SEQ ID NO. 5. In embodiments, thederivative has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleicacid sequence identity across the whole sequence or a portion of thesequence (e.g. a 5, 10, 20, 25 or more continuous nucleic acid portion)compared to SEQ ID NO. 5. Also, in embodiments, the derivative encodes afunctional derivative of TAT that has at least 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of theactivity of the TAT encoded by SEQ ID NO. 5.

In embodiments, the TAT peptide has the sequence of SEQ ID NO. 14 or aderivative thereof. In embodiments, the derivative has at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity to the sequence of SEQ ID NO. 14. Inembodiments, the derivative has at least 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity across the whole sequence or a portion of thesequence (e.g. a 5, 6, 7, 8, 9, 10, or more continuous amino acidportion) compared to SEQ ID NO. 14. Also, in embodiments, the derivativeis a functional derivative of the TAT peptide of SEQ ID NO. 14 that hasat least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98% or 99% of the activity of the polypeptide of SEQID NO. 14. In embodiments, the derivative has one or more conservativemodifications of SEQ ID NO. 14 (e.g., 1, 2, 3, 4, 5, 10, or moreconservative modifications).

A nuclear localization signal or sequence (NLS) is an amino acidsequence that tags a protein for import into the cell nucleus by nucleartransport. Any peptides that are known to be capable of NLS or havenuclear localization activity can be used in the composition and methodsof the disclosures. In embodiments, the recombinant protein has one ormore NLSs. In embodiments, the number of NLS present in the recombinantpeptide can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In embodiments,the NLS used in the disclosure is encoded by the sequence of SEQ ID NO.6 or a derivative thereof. In embodiments, the derivative has at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% of the nucleic acid sequence identity to thesequence of SEQ ID NO. 6. In embodiments, the derivative has at least90%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identityacross the whole sequence or a portion of the sequence (e.g. a 2, 3, 4,5 or more continuous nucleic acid portion) compared to SEQ ID NO. 6.Also, in embodiments, the derivative encodes a functional derivative ofNLS that has at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the activity of the NLSencoded by SEQ ID NO. 6.

In embodiments, the NLS has the sequence of SEQ ID NO: 15 or aderivative thereof. In embodiments, the derivative has at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity to the sequence of SEQ ID NO. 15. Inembodiments, the derivative has at least 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity across the whole sequence or a portion of thesequence (e.g. a 5, 6, 7, 8, 9, 10, or more continuous amino acidportion) compared to SEQ ID NO. 15. Also, in embodiments, the derivativeis a functional derivative of the NLS of SEQ ID NO. 15 that has at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% of the activity of the polypeptide of SEQ ID NO.15. In embodiments, the derivative has one or more conservativemodifications of SEQ ID NO. 15 (e.g., 1, 2, 3, 4, 5, 10, or moreconservative modifications).

In embodiments, the recombinant peptide of the present disclosure hasone or more additional sequences such as a myc-tag sequence andmaltose-binding sequence. A myc tag is a polypeptide protein tag derivedfrom the c-myc gene product that can be added to a protein usingrecombinant DNA technology. It can be used for affinity chromatography,then used to separate recombinant protein expressed by the hostorganism. It can also be used in the isolation of protein complexes withmultiple subunits. In embodiments, the recombinant peptide has a myc-tagsequence that is encoded by the sequence of SEQ ID NO. 7 or derivativethereof having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or99% of nucleic acid sequence identity to the sequence of SEQ ID NO. 7.In embodiments, the derivative has at least 90%, 95%, 96%, 97%, 98%, 99%or 100% nucleic acid sequence identity across the whole sequence or aportion of the sequence (e.g. a 2, 3, 4, 5 or more continuous nucleicacid portion) compared to SEQ ID NO. 7. Maltose binding peptide (MBP),which was originally found as an Escherichia coli gene and productthereof, can be used to increase the solubility of recombinant proteins.In this system, the protein of interest can be expressed as a MBP-fusionprotein, preventing aggregation of the protein of interest. In addition,MBP can also be used as an affinity tag for purification of recombinantproteins. Thus, the MBP-protein fusion can be purified by eluting thecolumn with maltose. Once the fusion protein is obtained in purifiedform, the protein of interest can often be cleaved from MBP with aspecific protease. The protein of interest can then be separated fromMBP by affinity chromatography. In embodiments, the recombinant peptidehas a MBP sequence that is known in the art or a derivative thereof.

In embodiments, the myc-tag has the sequence of SEQ ID NO. 16 or aderivative thereof. In embodiments, the derivative has at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity to the sequence of SEQ ID NO. 16. Inembodiments, the derivative has at least 90%, 95%, 96%, 97%, 98%, 99% or100% sequence identity across the whole sequence or a portion of thesequence (e.g. a 5, 6, 7, 8, 9, or 10 continuous amino acid portion)compared to SEQ ID NO. 16. Also, in embodiments, the derivative is afunctional derivative of the myc-tag of SEQ ID NO. 16 that has at least30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% of the activity of the polypeptide of SEQ ID NO.16. In embodiments, the derivative has one or more conservativemodifications of SEQ ID NO. 16 (e.g., 1, 2, 3, 4, 5, or moreconservative modifications).

In embodiments, the recombinant peptide of the present disclosure hasone or more of the following components: (i) a zinc finger domain (e.g.ZFP-362), (ii) a peptide capable of transcription activation (e.g. atranscription activator), (iii) a cell-penetrating sequence, (iv)nuclear localization sequence and (v) additional sequence for proteinstabilization and isolation (or purification). In embodiments, thenumber of each of the foregoing components present in a single moleculeof the recombinant peptide, independently, can be 0, 1, 2, 3, 4, 5, 6,7, 8, 9, 10 or more.

In embodiments, the recombinant peptide of the present disclosure has(i) a zinc finger domain (e.g. ZFP-362) and (ii) a peptide capable oftranscription activation (e.g. a transcription activator).

In embodiments, the recombinant peptide of the present disclosure has(i) a zinc finger domain (e.g. ZFP-362), (ii) a peptide capable oftranscription activation (e.g. a transcription activator) and (iii) acell-penetrating sequence.

In embodiments, the recombinant peptide of the present disclosure has(i) a zinc finger domain (e.g. ZFP-362), (ii) a peptide capable oftranscription activation (e.g. a transcription activator) and (iv)nuclear localization sequence.

In embodiments, the recombinant peptide of the present disclosure has(i) a zinc finger domain (e.g. ZFP-362), (ii) a peptide capable oftranscription activation (e.g. a transcription activator) and (v)additional sequence for protein stabilization and isolation (orpurification).

In embodiments, the recombinant peptide of the present disclosure has(i) a zinc finger domain (e.g. ZFP-362), (ii) a peptide capable oftranscription activation (e.g. a transcription activator), (iii) acell-penetrating sequence and (iv) nuclear localization sequence.

In embodiments, the recombinant peptide of the present disclosure has(i) a zinc finger domain (e.g. ZFP-362), (ii) a peptide capable oftranscription activation (e.g. a transcription activator), (iii) acell-penetrating sequence and (v) additional sequence for proteinstabilization and isolation (or purification).

In embodiments, the recombinant peptide of the present disclosure has(i) a zinc finger domain (e.g. ZFP-362), (ii) a peptide capable oftranscription activation (e.g. a transcription activator), (iv) nuclearlocalization sequence and (v) additional sequence for proteinstabilization and isolation (or purification). In some of theseembodiments, the recombinant peptide has the ZFP-362 as (i), a VPR as(ii), a NLS as (iv) and a myc-tag sequence as (v) as provided in SEQ IDNO. 8. In embodiments, the recombinant polypeptide has the sequence ofSEQ ID NO. 17, or a derivative thereof.

In embodiments, the recombinant peptide of the present disclosure has(i) a zinc finger domain (e.g. ZFP-362), (ii) a peptide capable oftranscription activation, (iii) a cell-penetrating sequence, (iv)nuclear localization sequence and (v) additional sequence for proteinstabilization and isolation (or purification). In some of theseembodiments, the recombinant peptide has the ZFP-362 as (i), a VPR as(ii), a TAT as (iii), a NLS as (iv) and a myc-tag sequence as (v) asprovided in SEQ ID NO. 9. In embodiments, the recombinant polypeptidehas the sequence of SEQ ID NO: 18, or a derivative thereof.

In another aspect, provided herein is a nucleotide sequence encoding anyof the recombinant peptides of the present disclosure.

In embodiments, the nucleotide sequence of the present disclosure has anucleotide sequence encoding a zinc finger domain. In embodiments, thenucleotide sequence of the present disclosure has the sequence of SEQ IDNO. 3 or a derivative thereof. In embodiments, the nucleotide sequenceof the present disclosure has a nucleotide sequence encoding the ZFP-362or a derivative thereof. In embodiments, the ZFP-362 has the sequence ofSEQ ID NO. 12, or a derivative thereof.

In embodiments, the nucleotide sequence of the present disclosure has anucleotide sequence encoding a peptide capable of transcriptionalactivation. In embodiments, the nucleotide sequence of the presentdisclosure has a nucleotide sequence encoding a transcriptionalactivator. In embodiments, the nucleotide sequence of the presentdisclosure has the sequence of SEQ ID NO.4 or a derivative thereof. Inembodiments, the nucleotide sequence has a nucleotide sequence encodinga VPR or a derivative thereof. In embodiments, the VPR has the sequenceof SEQ ID NO. 13 or a derivative thereof. In embodiments, instead of VPRor in combination with VPR, one or more of known transcriptionactivators such as p65 transactivating subunit of NF-kappa B, heat-shockfactor 1 (HSF) activation domain, the VP64 (tetramer of VP16) activationdomain, synergistic activation mediator (SAM) and any derivativesthereof can be encoded by the nucleotide sequence of the disclosures.

In embodiments, the nucleotide sequence of the present disclosure has anucleotide sequence encoding a cell-penetrating sequence. Inembodiments, the nucleotide sequence of the present disclosure has thesequence of SEQ ID NO.5 or a derivative thereof. In embodiments, thenucleotide sequence has a nucleotide sequence encoding a TAT or aderivative thereof. In embodiments, the TAT has the sequence of SEQ IDNO: 14 or a derivative thereof.

In embodiments, the nucleotide sequence of the present disclosure has anucleotide sequence encoding a nuclear localization sequence. Inembodiments, the nucleotide sequence of the present disclosure has thesequence of SEQ ID NO.6 or a derivative thereof. In embodiments, thenucleotide sequence has a nucleotide sequence encoding an NLS. Inembodiments, the NLS has the sequence of SEQ ID NO. 15 or a derivativethereof.

In embodiments, the nucleotide sequence of the present disclosure has anucleotide sequence encoding an additional sequence for proteinstabilization and isolation (or purification). In embodiments, thenucleotide sequence of the present disclosure has the sequence of SEQ IDNO.7 or a derivative thereof. In embodiments, the nucleotide sequencehas a nucleotide sequence encoding a myc-tag sequence or a maltosebinding peptide (MBP). In embodiments, the myc-tag has the sequence ofSEQ ID NO. 16 or a derivative thereof. In embodiments, the nucleotidesequence of the present disclosure has nucleic acid sequence(s) encodingan additional peptide sequence for protein stabilization and isolation(or purification); however, the resulting recombinant peptide does nothave such an additional peptide sequence as the additional peptidesequence can be removed during an intermediate isolation and/orpurification process

In embodiments, the nucleotide sequence of the present disclosure hasone or more sequences, each of which encodes one of the followingcomponents: (i) a zinc finger domain (e.g. ZFP-362), (ii) a peptidecapable of transcription activation (e.g. a transcription activator),(iii) a cell-penetrating sequence, (iv) nuclear localization sequenceand (v) additional sequence for protein stabilization and isolation (orpurification). In embodiments, the number of each sequence encoding oneof the foregoing components that is present in a single molecule of thenucleotide sequence, independently, is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more. Also, any nucleic acid in the nucleotide sequence of thedisclosures can be a natural or non-natural nucleic acid, e.g. modifiednucleic acid.

In embodiments, the nucleotide sequence of the present disclosure hassequences encoding (i) a zinc finger domain (e.g. ZFP-362) and (ii) apeptide capable of transcription activation (e.g. a transcriptionactivator).

In embodiments, the nucleotide sequence of the present disclosure hassequences encoding (i) a zinc finger domain (e.g. ZFP-362), (ii) apeptide capable of transcription activation (e.g. a transcriptionactivator) and (iii) a cell-penetrating sequence.

In embodiments, the nucleotide sequence of the present disclosure hassequences encoding (i) a zinc finger domain (e.g. ZFP-362), (ii) apeptide capable of transcription activation (e.g. a transcriptionactivator) and (iv) nuclear localization sequence.

In embodiments, the nucleotide sequence of the present disclosure hassequences encoding (i) a zinc finger domain (e.g. ZFP-362), (ii) apeptide capable of transcription activation (e.g. a transcriptionactivator) and (v) additional sequence for protein stabilization andisolation (or purification).

In embodiments, the nucleotide sequence of the present disclosure hassequences encoding (i) a zinc finger domain (e.g. ZFP-362), (ii) apeptide capable of transcription activation (e.g. a transcriptionactivator), (iii) a cell-penetrating sequence and (iv) nuclearlocalization sequence.

In embodiments, the nucleotide sequence of the present disclosure hassequences encoding (i) a zinc finger domain (e.g. ZFP-362), (ii) apeptide capable of transcription activation (e.g. a transcriptionactivator), (iii) a cell-penetrating sequence and (v) additionalsequence for protein stabilization and isolation (or purification).

In embodiments, the nucleotide sequence of the present disclosure hassequences encoding (i) a zinc finger domain (e.g. ZFP-362), (ii) apeptide capable of transcription activation (e.g. a transcriptionactivator), (iv) nuclear localization sequence and (v) additionalsequence for protein stabilization and isolation (or purification). Insome of these embodiments, the nucleotide sequence has sequencesencoding the ZFP-362 as (i), a VPR as (ii), a NLS as (iv) and a myc-tagsequence as (v) as provided in SEQ ID NO. 8. In embodiments, thenucleotide sequence encodes a protein having the sequence of SEQ ID NO.17 or a derivative thereof.

In embodiments, the nucleotide sequence of the present disclosure hassequences encoding (i) a zinc finger domain (e.g. ZFP-362), (ii) apeptide capable of transcription activation, (iii) a cell-penetratingsequence, (iv) nuclear localization sequence and (v) additional sequencefor protein stabilization and isolation (or purification). In some ofthese embodiments, the nucleotide sequence has sequences encoding theZFP-362 as (i), a VPR as (ii), a TAT as (iii), a NLS as (iv) and amyc-tag sequence as (v) as provided in SEQ ID NO. 9. In embodiments, thenucleotide sequence encodes a protein having the sequence of SEQ ID NO.18 or a derivative thereof.

In one aspect, provided is a vector such as an expression vector thathas any of the nucleotide sequences of the present disclosure, whichencode the recombinant peptide of the present disclosure. Therefore, inembodiments the expression vector can be used to produce the recombinantpeptide of the disclosures in cells. The expression vector can betransfected into cells (e.g. eukaryotic cells such as mammalian cells orhuman cell lines or prokaryotic cells such as Escherichia coli, in whichthe recombinant peptide is expressed and the expressed peptide can beisolated and purified using various techniques available in the field.

In embodiments, the expression vector is capable of directing theexpression of nucleic acids to which they are operatively linked. Theterm “operably linked” means that the nucleotide sequence of interest islinked to regulatory sequence(s) in a manner that allows for expressionof the nucleotide sequence. The regulatory sequence may include, forexample, promoters, enhancers and other expression control elements(e.g., polyadenylation signals). Such regulatory sequences are wellknown in the art and are described, for example, in Goeddel; GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990). Regulatory sequences include those that directconstitutive expression of a nucleotide sequence in many types of hostcells, and those that direct expression of the nucleotide sequence onlyin certain host cells (e.g., tissue-specific regulatory sequences). Itwill be appreciated by those skilled in the art that the design of theexpression vector can depend on such factors as the choice of the targetcell, the level of expression desired, and the like.

Expression vectors contemplated include, but are not limited to, viralvectors based on various viral sequences as well as those contemplatedfor eukaryotic target cells or prokaryotic target cells. The “targetcells” may refer to the cells where the expression vector is transfectedand the nucleotide sequence encoding the recombinant peptide isexpressed. In embodiments, the target cells are the cells used forproduction of the recombinant peptide of the present disclosure forlater use. Therefore, in these embodiments the expressed recombinantpeptides are isolated from the target cells and administered to asubject later for a therapeutic purpose, e.g. treatment of HIV in thesubject. In alternative embodiments, the target cells may refer to thecells that have latent HIV. Therefore, when the expression vector istransfected into such cells, the recombinant peptide expressed from thevector activates the viral transcription in the cells, activating latentHIV in the target cells. Any vectors can be used so long as they arecompatible with the desired or intended target cell. The skilled personin the art can use any suitable vectors known and available in the artdepending on their system, e.g. the target cell or the process ofculturing cell and purifying the recombinant peptides.

In some examples, a vector has one or more transcription and/ortranslation control elements. Depending on the target/vector systemutilized, any of a number of suitable transcription and translationcontrol elements, including constitutive and inducible promoters,transcription enhancer elements, transcription terminators, etc. can beused in the expression vector.

Non-limiting examples of suitable eukaryotic promoters (i.e., promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, H1, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, humanelongation factor-1 promoter (EF1), a hybrid construct having thecytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter(CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1locus promoter (PGK), and mouse metallothionein-I. The promoter can be aconstitutive promoter (e.g., CMV promoter, UBC promoter). In some cases,the promoter can be a spatially restricted and/or temporally restrictedpromoter (e.g., a tissue specific promoter, a cell type specificpromoter, etc.).

In embodiments, the expression vector has sequences encoding one or moreof the following components: (i) a zinc finger domain (e.g. ZFP-362),(ii) a peptide capable of transcription activation (e.g. a transcriptionactivator), (iii) a cell-penetrating sequence, (iv) nuclear localizationsequence and (v) additional sequence for protein stabilization andisolation (or purification). In embodiments, the number of each sequenceencoding one of the foregoing components that is present in a singleexpression vector, independently, is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore.

In one example, the expression vector has sequences encoding the ZFP-362as (i), a VPR as (ii), a TAT as (iii), three NLSs as (iv) and a MBP as(v) as illustrated in FIG. 2 . The vector of FIG. 2 has the nucleotidesequence encoding a fusion peptide of the ZFP-362, targeting to theNF-κB doublet in the HIV LTR and the VPR transcriptional activatordomain (VP64+RelA (p65) and Rta AD) which is expressed from the CMVpromoter. This vector further has a sequence encoding a MBP with afactor Xa cleavage for purification of recombinant peptide, a TATpeptide for nuclear targeting and transit of the recombinant peptidethrough the blood brain barrier and three nucleoplasmin nuclearlocalization signals (NLS) for enhanced nuclear targeting of therecombinant peptide. The entire recombinant peptide expressed from thisvector is terminated by the bGH poly A signal.

Methods

In one aspect, provided is a method of activating a latent HIV from acell. The method can have administering any of the recombinant peptidesor any of the expression vectors disclosed herein to the cell.

In embodiments, the cell is a cell in which HIV is in the latency. Thismeans that the cell was previously infected with HIV and the HIV orprogeny thereof become latent in the cell, e.g. in the episomal orproviral latency. The recombinant peptide of the disclosures may have azinc finger domain that specifically recognizes and binds to a sequence(i.e. a target sequence) in the HIV LTR. The recombinant peptide mayalso have a peptide capable of transcription activation such that whenthe recombinant peptide is bound to the target sequence, it can promotetranscription of the HIV LRT sequence, which leads to the transcriptionof the viral genome. Therefore, when the recombinant peptide is providedinto the infected cell with latent HIV, the latent HIV is activated andno longer in its latency due to the activity of the recombinant peptide.

In embodiments, the cell having the viral genome in its own genome (i.e.proviral latency) or as a separate nucleic acid molecule (i.e. episomallatency) is T cell, macrophage, monocyte or microglial cell. Therefore,in embodiments a population of cells having one or more of T cell,macrophage, monocyte and microglial cell that are infected with latentHIV is subjected to the compositions and methods of the presentdisclosure.

In embodiments, in order to activate latent HIV from the infected cell,the recombinant peptide of the disclosures is provided to the cell.Alternatively, any of the expression vector of the disclosures thatencodes the recombinant peptide can be provided to the cell, expressingthe recombinant peptide and exhibiting the desired activity, i.e.activation of latent HIV. This provision (or delivery) of therecombinant peptide or the expression vector thereof to the infectedcell can be done using various techniques available in the art, whichinclude, but not limited to, alcium phosphate transfection, liposomaltransfection, nucleofection, sonoporation, transfection through heatshock, magnetifection and electroporation.

In one aspect, provided is a method of treating HIV in a subject in needthereof. The method may have administering any of the recombinantpeptides or any of the expression vectors disclosed herein to thesubject.

In embodiments, the subject has one or more cells in which the HIV orprogeny thereof is in a latent stage. In embodiments, the latent HIVpresent in certain cells of the subject is in episomal latency orproviral latency. In embodiments, the infected cells with the latent HIVin the subject can be one or more of T cell, macrophage, monocyte, andmicroglial cell.

In embodiments, the treatment method of the present disclosure also hasselectively killing the infected cells in which the latent HIV isactivated by the recombinant peptide.

In embodiments, the method of treating HIV has administering apharmaceutical composition or formulation to a subject in need of thetreatment. In embodiments, the pharmaceutical compositions andformulations for treating HIV, in particular latent HIV in the subject,have compounds in accordance with the present disclosure (e.g. therecombinant peptide, the nucleic acid encoding the recombinant peptideand/or the expression vector expressing the recombinant peptide).

In embodiments, the pharmaceutical composition has one or more compoundsof the present disclosure and one or more pharmaceutically acceptableexcipients. In embodiments, the pharmaceutical compositions, thecompound, or pharmaceutically acceptable salt thereof, is included in atherapeutically effective amount.

The pharmaceutical composition of the present disclosure can be preparedand administered in a wide variety of dosage formulations. Compoundsdescribed can be administered orally, rectally, or by injection (e.g.intravenously, intramuscularly, intracutaneously, subcutaneously,intraduodenally, or intraperitoneally). For example, the compositionsdisclosed herein can be delivered by transdermally, by a topical route,formulated as applicator sticks, solutions, suspensions, emulsions,gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.Oral preparations include tablets, pills, powder, dragees, capsules,liquids, lozenges, cachets, gels, syrups, slurries, suspensions, etc.,suitable for ingestion by the patient. Solid form preparations includepowders, tablets, pills, capsules, cachets, suppositories, anddispersible granules. Liquid form preparations include solutions,suspensions, and emulsions, for example, water or water/propylene glycolsolutions. The compositions of the present disclosure can additionallyinclude components to provide sustained release and/or comfort. Suchcomponents include high molecular weight, anionic mucomimetic polymers,gelling polysaccharides and finely-divided drug carrier substrates.These components are discussed in greater detail in U.S. Pat. Nos.4,911,920; 5,403,841; 5,212,162; and 4,861,760. The entire contents ofthese patents are incorporated herein by reference in their entirety forall purposes. The compositions disclosed herein can also be delivered asmicrospheres for slow release in the body. For example, microspheres canbe administered via intradermal injection of drug-containingmicrospheres, which slowly release subcutaneously (see Rao, J. BiomaterSci. Polym. Ed. 7:623-645, 1995; as biodegradable and injectable gelformulations (see, e.g., Gao Pharm. Res. 12:857-863, 1995); or, asmicrospheres for oral administration (see, e.g., Eyles, J. Pharm.Pharmacol. 49:669-674, 1997). In another embodiment, the formulations ofthe compositions of the present disclosure can be delivered by the useof liposomes which fuse with the cellular membrane or are endocytosed,i.e., by employing receptor ligands attached to the liposome, that bindto surface membrane protein receptors of the cell resulting inendocytosis. By using liposomes, particularly where the liposome surfacecarries receptor ligands specific for target cells, or are otherwisepreferentially directed to a specific organ, one can focus the deliveryof the compositions of the present disclosure into the target cells invivo. (See, e.g., Al-Muhammed, J. Microencapsul. 13:293-306, 1996;Chonn, Curr. Opin. Biotechnol. 6:698-708, 1995; Ostro, Am. J. Hosp.Pharm. 46:1576-1587, 1989). The compositions can also be delivered asnanoparticles.

Pharmaceutical compositions can include compositions wherein the activeingredient (e.g. compounds described herein, including embodiments orexamples) is contained in a therapeutically effective amount, i.e., inan amount effective to achieve its intended purpose. The actual amounteffective for a particular application will depend, inter alia, on thecondition being treated. When administered in methods to treat adisease, such compositions will contain an amount of active ingredienteffective to achieve the desired result, e.g., modulating the activityof a target molecule, in particular activation of latent HIV from cellspresent in the subject who was treated with the composition.

In embodiments, the effective amount is an amount sufficient toaccomplish a stated purpose (e.g. achieve the effect for which it isadministered, treat a disease, reduce the number of cells infected withlatent HIV, reduce one or more symptoms of a disease or condition). Anexample of the effective amount is an amount sufficient to contribute tothe desired treatment such as activation of latent HIV from the infectedcells that would be sufficient to completely or substantially eradicatethe HIV infection from the subject. This amount can also be referred toas a therapeutically effective amount. Thus, in some examples, for thegiven parameter, an effective amount will show activation of latent HIVfrom at least 5%, 10%, 15%, 20%, 25%, 40%, 50%, 60%, 75%, 80%, 90%, orat least 100% of the total number of infected cells that are infectedwith latent HIV. The exact amounts will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques.

In embodiments, the effective amount of the composition in accordancewith the present disclosure or active ingredient thereof, e.g. therecombinant peptide, the nucleic acid encoding the recombinant peptideand/or the expression vector expressing the recombinant peptide, isadministered to a subject in need thereof. In embodiments, the effectiveamount of the composition or active ingredient thereof to beadministered to the subject in one application is about 1 ng/kg ofsubject body weight, about 10 ng/kg of subject body weight, about 50ng/kg of subject body weight, about 100 ng/kg of subject body weight,about 500 ng/kg of subject body weight, about 1 μg/kg of subject bodyweight, about 10 μg/kg of subject body weight, about 50 μg/kg of subjectbody weight, about 100 μg/kg of subject body weight, about 150 μg/kg ofsubject body weight, about 200 μg/kg of subject body weight, about 250μg/kg of subject body weight, about 300 μg/kg of subject body weight,about 350 μg/kg of subject body weight, about 375 μg/kg of subject bodyweight, about 400 μg/kg of subject body weight, about 450 μg/kg ofsubject body weight, about 500 μg/kg of subject body weight, about 550μg/kg of subject body weight, about 600 μg/kg of subject body weight,about 650 μg/kg of subject body weight, about 700 μg/kg of subject bodyweight, about 750 μg/kg of subject body weight, about 800 μg/kg ofsubject body weight, about 850 μg/kg of subject body weight, about 900μg/kg of subject body weight, about 1 mg/kg of subject body weight,about 10 mg/kg of subject body weight, about 50 mg/kg of subject bodyweight, about 100 mg/kg of subject body weight, about 500 mg/kg ofsubject body weight, about 1 g/kg of subject body weight or more or anyintervening ranges of the of the foregoing. In embodiments, theeffective amount of the composition or active ingredient thereof to beadministered to the subject in one application is about 0.5 μg, about1.0 μg, about 1.5 μg, about 2.0 μg, about 2.5 μg, about 3.0 μg, about3.5 μg, about 4.0 μg, about 4.5 μg about 5.0 μg, about 5.5 μg, about 6.0μg, about 6.5 μg, about 7.0 μg, about 7.5 μg, about 8.0 μg, about 8.5μg, about 9.0 μg, about 9.5 μg, about 1.0 mg, about 1.5 mg, about 2.0mg, about 2.5 mg, about 3.0 mg, about 3.5 mg, about 4.0 mg, about 4.5 mgabout 5.0 mg, about 5.5 mg, about 6.0 mg, about 6.5 mg, about 7.0 mg,about 7.5 mg, about 8.0 mg, about 8.5 mg, about 9.0 mg, about 9.5 mg,about 1 g or more or any intervening ranges of the foregoing. Inembodiments, one or more than one applications of the compositioncontaining the active ingredient can be administered to the subject overa period of time, e.g. several hours, several days, several weeks orseveral months.

The dosage and frequency (single or multiple doses) administered to amammal can vary depending upon a variety of factors, for example,whether the mammal suffers from another disease, and its route ofadministration; size, age, sex, health, body weight, body mass index,and diet of the recipient; nature and extent of symptoms of the diseasebeing treated, kind of concurrent treatment, complications from thedisease being treated or other health-related problems. Adjustment andmanipulation of established dosages (e.g., frequency and duration) arewell within the ability of those skilled in the art.

In embodiments, the compounds described herein can be used as a soleactive ingredient(s) of a composition. In embodiments, the compounds canbe used in combination with one another, or with other active compoundsor drugs known to be useful in treating HIV or with adjunctive agentsthat may not be effective alone, but may contribute to the efficacy ofthe active agent. In embodiments, the composition further has one ormore other active compounds or drugs known to be useful in treating HIVor cells infected with HIV. In embodiments, the compounds describedherein may be co-administered with one another or with one or more otheractive compounds or drugs known to be useful in treating HIV or cellsinfected with HIV that are known in the field.

In embodiments, non-limiting examples of the drugs to treat HIV that canbe co-administered with the composition of the present disclosureinclude, but not limited to, efavirenz, emtricitabine, tenofovirdisoproxil fumarate rilpivirine, elvitegravir, cobicistat, abacavir,dolutegravir, lamivudine, zidovudine, didanosine, stavudine etravirine,delavirdine mesylate, nevirapine, tipranavir, indinavir, atazanavir,saquinavir, lopinavir, ritonavir, fosamprenavir, darunavir, nelfinavir,simeprevir, boceprevir, ombitasvir, paritaprevir, dasabuvir,enfuvirtide, raltegravir, dolutegravir, elvitegravir, maraviroc or anycombinations thereof.

For preparing pharmaceutical compositions from compounds describedherein, pharmaceutically acceptable carriers can be either solid orliquid. Solid form preparations include powders, tablets, pills,capsules, cachets, suppositories, and dispersible granules. A solidcarrier may be one or more substance that may also act as diluents,flavoring agents, binders, preservatives, tablet disintegrating agents,or an encapsulating material.

Utilizing the teachings provided herein, an effective therapeutictreatment regimen can be planned that does not cause substantialtoxicity and yet is entirely effective to treat the clinical symptomsdemonstrated by the particular patient. This planning should involve thecareful choice of active compound by considering factors such ascompound potency, relative bioavailability, patient body weight,presence and severity of adverse side effects, preferred mode ofadministration, and the toxicity profile of the selected agent.

Kits

In one aspect, provided herein is a kit for, in part, activation oflatent HIV from infected cells and treatment of HIV. As part of the kit,materials and instruction are provided for both the activation of thelatent virus in the infected cells present in a subject, e.g. a patientand the preparation of reaction mixtures for storage and use of kitcomponents.

In embodiments, the kit can contain one or more of the followingcomponents:

1. any of the recombinant peptides in the present disclosure,

2. any of the nucleic acid sequences disclosed herein which encodes anyof the recombinant peptides,

3. any of the expression vectors disclosed herein which expresses any ofthe recombinant peptides, and

4. instructions for how to use the kit components.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EXAMPLES Example 1: 362-ZFP-VPR-TAT Transcriptional Activation of LatentHIV

We have developed a therapeutic compound (a unique recombinant protein)that can specifically target and sustainably activate HIV expressionfrom latently infected cells. Using well-established freely availablealgorithms (ZF Tools Ver 3.0), we developed and screened three zincfinger proteins (ZFPs) and found 1 ZFP, ZFP-362 (FIG. 1A and FIG. 2 ),that specifically targets the HIV-1 LTR (promoter) and inducesactivation of HIV transcription at levels comparable to defective CRISPRVPR conjugates (dCas-VPR) (FIGS. 1B-1C), the gold standard in activationof latent HIV. The ZFP-362-VPR targeted activation of HIV LTR expressionis specifically targeted to the well-defined NF-κB doublet site, aregion known to be susceptible to modulation and control of viraltranscription and only found in HIV, as targeting of ZFP-362-VPR tocells lacking this site are not activated (FIG. 1D) compared with thosecontaining this site (FIG. 1C). Moreover, CHIP analysis demonstratedthat ZFP-362-VPR binds to the 5′ LTR along with defective CRISPRtargeting as a control (FIG. 3 ). Lastly, the recombinant form ofZFP-362-VPR was functional in recombinant protein form when contrastedwith control crude-lysates (FIG. 4 ).

We have developed the Tat peptide conjugate as part of the ZFP-362-VPRdescribed here (FIG. 1A). This recombinant protein ZFP-362-VPR (FIG. 2), which contains the Tat peptide domain as well as various nuclearlocalization signals (NLS) and a cleavable Maltose binding domain, isfunctional as a protein administered directly to cells or infectedpatients. The Tat peptide domain facilitates transfer to the nucleus ofcells as well as across the blood brain barrier and the ZFP-362 providesthe specificity in targeting the NF-κB doublet that is only found in theHIV LTR. Once the LTR is targeted the VPR induces activation of thelatent provirus, resulting in activation of latent reservoirs of HIV.

Example 2: Efficacy of the Recombinant ZFP-362-VPR Peptide

Cell culture. The cell lines were maintained in Dulbecco's modifiedEagle medium (DMEM, Thermo Scientific, MA, USA) and 10% fetal bovineserum (FBS). The HEK293-GP160 (92UG037.8) cell line was used and themedia was supplemented with 1.5 ug/ml puromycin. The cell lines werecultured at 37° C. with 5% CO₂.

Transfection of cells. ZFP-362 and gRNAs with dCas-VPR as well ascontrols were transfected directly into pMo or LCHiT cells (˜1 μg ofplasmid total/10⁶ cells) using Lipofectamine Max or Neon Electroporation(Invitrogen, Carlsbad, Calif.). Expression of GFP (pMo-GFP) or mCherry(LChIT CEM cells) was determined using FACS and qRTPCR for GFP ormCherry expression standardized to Beta Actin or GAPDH.

CHIP analysis of ZFP-362-VPR binding the 5′ LTR. In order to determinethe direct binding of ZFP-362-VPR to the proviral LTR a chromatinimmunoprecipitation assay (ChIP) was carried out using Anti-Myc tagantibody (ab9106) with 5′ LTR primer 5′-TTTCCGCTGGGGACTTTCCAG-3′ (SEQ IDNO:10) and 3′ LTR primer 5′-ACTCAAGGCAAGCTTTATTGAGGC-3′(FIG. 3 ; SEQ IDNO:11). The no antibody background is subtracted from the sampletreatment and controls and input and the treatment and controls are thenstandardized to the input.

Top 10 off-target promoter loci bound by ZFP-362-VPR (Table 1) and gRNAF2-dCas-VPR (Table 2) were tested. Triplicate treated cells were treatedand 72 hrs later CHiP performed with Anti-Myc tag antibody Abcam(ab9132) followed by High throughput deep sequencing. The results belowshow that the ZFP-362-VPR is very on-target, as good as CRISPR if notbetter at on targeting.

FIG. 6 shows Table 1: Top ZFP-362-VPR off-target gene promoter boundsites.

FIG. 7 shows Table 2: Top F2-gRNA-362-dCasVPR off-target gene promoterbound sites.

Example 3: Activation of Various HIV-1 Subtypes

HEK293 cells were transfected in triplicate using Lipofectamine 3000®(Thermo Fisher scientific, Mass., USA) with pcDNA-ZFP-362-VPR orpcDNA-VEGF-VPR, along with vectors expressing firefly luciferase off theLTRs from different subtypes of HIV (Cat. No. 4787, 4788, 4789, 4790,4791, 4792, 4793; reagents were obtained through the NIH AIDS ReagentProgram, Division of AIDS, NIAID, NIH: pBlue3′LTR-luc-A from Dr. ReinkJeeninga and Dr. Ben Berkhout (Jeeninga et al., J. Virol. 74:3740-3751,2000; Klave and Berkhout, J. Virol. 68:3830-3840, 1994)). A vectorexpressing Renilla luciferase was included as a background control(pRL-CMV, Promega, Wis., USA). At 48 hrs post-transfection, aDual-luciferase® Reporter Assay was performed according tomanufacturer's instructions and luciferase activity detected on theGlomax® Explorer system (Promega, Wis., USA). The levels of fireflyluciferase were normalized to Renilla luciferase, and made relative tothe pcDNA-VEGF-VPR control. Results are depicted graphically in FIG. 8 .

Sequences around the LTR-362 site (SEQ ID NO:1) targeted by ZFP-362 ineach of the various subtypes were aligned, and the alignments areillustrated in FIG. 9 . Two possible binding sites for subtype C areillustrated. However, of the two sites, SEQ ID NO: 1 is more homologousto the upstream NF-κB III site in subtype C.

REFERENCES

-   1. Saayman S M, Lazar D C, Scott T A, Hart J R, Takahashi M, Burnett    J C, Planelles V, Morris K V, Weinberg M S. Potent and Targeted    Activation of Latent HIV-1 Using the CRISPR/dCas9 Activator Complex.    Mol. Ther. 2016; 24(3):488-98. doi: 10.1038/mt.2015.202. PubMed    PMID: 26581162; PMCID: PMC4786915.-   2. Bailus B J, Pyles B, McAlister M M, O'Geen H, Lockwood S H, Adams    A N, Nguyen J T, Yu A, Berman R F, Segal D J. Protein Delivery of an    Artificial Transcription Factor Restores Widespread Ube3a Expression    in an Angelman Syndrome Mouse Brain. Mol. Ther. 2016; 24(3):548-55.    doi: 10.1038/mt.2015.236. PubMed PMID: 26727042; PMCID: PMC4786922.

INFORMAL SEQUENCE LISITNGSEQ ID NO: 1 (The LTR-362 binding site for ZFP-362):CTTTCCGCTGGGGACTTTCCASEQ ID NO: 2 (ZFP-362-binding site in the HIV LTR.):AATGAAGGAGAGAACAACAGCTTGTTACACCCTATGAGCCAGCATGGGATGGAGGACCCGGAGGGAGAAGTATTAGTGTGGAAGTTTGACAGCCTCCTAGCATTTCGTCACATGGCCCGAGAGCTGCATCCGGAGTACTACAAAGACTGCTGACATCGAGCTTTCTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGTGTGGCCTGGGCGGGACTGGGGAGTGGCGAGCCCTCAGATGCTACATATAAGCAGCTGCTTTTTGCCTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCT TAAGSEQ ID NO: 3 (ZFP-362-VPR-NLS. ZFP-362 sequence)CTCGAACCTGGGGAGAAACCCTACAAGTGCCCCGAATGCGGGAAAAGCTTCTCACGCAAGGACAATCTCAAGAATCACCAGCGGACGCACACCGGAGAGAAGCCCTACAAGTGCCCCGAATGCGGAAAATCATTCTCACAACGCGCCCACTTGGAACGCCACCAGAGAACACACACAGGGGAGAAGCCATACAAGTGCCCTGAATGCGGCAAGTCTTTCAGTGAAAGGTCCCATCTGCGAGAGCACCAGCGAACACATACTGGCGAGAAGCCGTACAAGTGTCCCGAGTGCGGCAAGAGTTTTAGTTCCAAAAAACACCTGGCCGAACATCAGCGGACTCACACAGGGGAGAAGCCCTATAAATGCCCCGAGTGCGGCAAGAGCTTTAGCGATCCCGGGGCCCTCGTCCGACATCAGAGGACCCACACAGGGGAGAAACCTTACAAGTGTCCTGAATGCGGCAAATCTTTCAGCCAGAGAGCAAACCTGCGAGCTCACCAGAGAACCCATACTGGCGAAAAGCCTTATAAATGCCCTGAATGCGGGAAGAGTTTCAGCCGCTCTGACCACCTGACTACTCACCAGCGGACACACACTGGGAAGAAAACTAGCAGCGCTGCTGACCCCAAGAAGAAGAGGAAGGTGSEQ ID NO: 4 (ZFP-362-VPR-NLS. VPR sequence)TCGCCAGGGATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGAAGTTCCGGATCTCCGAAAAAGAAACGCAAAGTTGGTAGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGAAGCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCACCATCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGCCGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATCCCTGTGGCCCCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAATGGACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGCCGATATGGATTTCTCAGCCTTGCTGGGCTCTGGCAGCGGCAGCCGGGATTCCAGGGAAGGGATGTTTTTGCCGAAGCCTGAGGCCGGCTCCGCTATTAGTGACGTGTTTGAGGGCCGCGAGGTGTGCCAGCCAAAACGAATCCGGCCATTTCATCCTCCAGGAAGTCCATGGGCCAACCGCCCACTCCCCGCCAGCCTCGCACCAACACCAACCGGTCCAGTACATGAGCCAGTCGGGTCACTGACCCCGGCACCAGTCCCTCAGCCACTGGATCCAGCGCCCGCAGTGACTCCCGAGGCCAGTCACCTGTTGGAGGATCCCGATGAAGAGACGAGCCAGGCTGTCAAAGCCCTTCGGGAGATGGCCGATACTGTGATTCCCCAGAAGGAAGAGGCTGCAATCTGTGGCCAAATGGACCTTTCCCATCCGCCCCCAAGGGGCCATCTGGATGAGCTGACAACCACACTTGAGTCCATGACCGAGGATCTGAACCTGGACTCACCCCTGACCCCGGAATTGAACGAGATTCTGGATACCTTCCTGAACGACGAGTGCCTCTTGCATGCCATGCATATCAGCACAGGACTGTCCATCTTCGACACATCTCTGTTT SEQ ID NO: 5 (ZFP-362-VPR-Tat. TAT sequence)GGCCGTAAAAAACGTCGTCAGCGCCGTCGCGTCGACCTTSEQ ID NO: 6 (ZFP-362-VPR-NLS. NLS sequence)AAGCGACCTGCCGCCACAAAGAAGGCTGGACAGGCTAAGAAGAAGAAASEQ ID NO: 7 (ZFP-362-VPR-NLS. Myc sequence)GAGCAGAAGCTGATCTCAGAGGAGGACCTGCTTSEQ ID NO: 8 (ZFP-362-VPR-NLS. The recombinant protein is Myc-NLS-ZFP-362-VPR, is shown below in a 5′-3′ manner.):GAGCAGAAGCTGATCTCAGAGGAGGACCTGCTTAAGCGACCTGCCGCCACAAAGAAGGCTGGACAGGCTAAGAAGAAGAAACTCGAACCTGGGGAGAAACCCTACAAGTGCCCCGAATGCGGGAAAAGCTTCTCACGCAAGGACAATCTCAAGAATCACCAGCGGACGCACACCGGAGAGAAGCCCTACAAGTGCCCCGAATGCGGAAAATCATTCTCACAACGCGCCCACTTGGAACGCCACCAGAGAACACACACAGGGGAGAAGCCATACAAGTGCCCTGAATGCGGCAAGTCTTTCAGTGAAAGGTCCCATCTGCGAGAGCACCAGCGAACACATACTGGCGAGAAGCCGTACAAGTGTCCCGAGTGCGGCAAGAGTTTTAGTTCCAAAAAACACCTGGCCGAACATCAGCGGACTCACACAGGGGAGAAGCCCTATAAATGCCCCGAGTGCGGCAAGAGCTTTAGCGATCCCGGGGCCCTCGTCCGACATCAGAGGACCCACACAGGGGAGAAACCTTACAAGTGTCCTGAATGCGGCAAATCTTTCAGCCAGAGAGCAAACCTGCGAGCTCACCAGAGAACCCATACTGGCGAAAAGCCTTATAAATGCCCTGAATGCGGGAAGAGTTTCAGCCGCTCTGACCACCTGACTACTCACCAGCGGACACACACTGGGAAGAAAACTAGCAGCGCTGCTGACCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGAAGTTCCGGATCTCCGAAAAAGAAACGCAAAGTTGGTAGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGAAGCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCACCATCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGCCGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATCCCTGTGGCCCCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAATGGACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGCCGATATGGATTTCTCAGCCTTGCTGGGCTCTGGCAGCGGCAGCCGGGATTCCAGGGAAGGGATGTTTTTGCCGAAGCCTGAGGCCGGCTCCGCTATTAGTGACGTGTTTGAGGGCCGCGAGGTGTGCCAGCCAAAACGAATCCGGCCATTTCATCCTCCAGGAAGTCCATGGGCCAACCGCCCACTCCCCGCCAGCCTCGCACCAACACCAACCGGTCCAGTACATGAGCCAGTCGGGTCACTGACCCCGGCACCAGTCCCTCAGCCACTGGATCCAGCGCCCGCAGTGACTCCCGAGGCCAGTCACCTGTTGGAGGATCCCGATGAAGAGACGAGCCAGGCTGTCAAAGCCCTTCGGGAGATGGCCGATACTGTGATTCCCCAGAAGGAAGAGGCTGCAATCTGTGGCCAAATGGACCTTTCCCATCCGCCCCCAAGGGGCCATCTGGATGAGCTGACAACCACACTTGAGTCCATGACCGAGGATCTGAACCTGGACTCACCCCTGACCCCGGAATTGAACGAGATTCTGGATACCTTCCTGAACGACGAGTGCCTCTTGCATGCCATGCATATCAGCACAGGACTGTCCATCTTCGACACATCTCTGTTTSEQ ID NO: 9 (ZFP-362-VPR-Tat. The recombinant protein is TAT-Myc-NLS-ZFP-362-VPR is shown below in a 5′-3′ manner.GGCCGTAAAAAACGTCGTCAGCGCCGTCGCGTCGACCTTGAGCAGAAGCTGATCTCAGAGGAGGACCTGCTTAAGCGACCTGCCGCCACAAAGAAGGCTGGACAGGCTAAGAAGAAGAAACTCGAACCTGGGGAGAAACCCTACAAGTGCCCCGAATGCGGGAAAAGCTTCTCACGCAAGGACAATCTCAAGAATCACCAGCGGACGCACACCGGAGAGAAGCCCTACAAGTGCCCCGAATGCGGAAAATCATTCTCACAACGCGCCCACTTGGAACGCCACCAGAGAACACACACAGGGGAGAAGCCATACAAGTGCCCTGAATGCGGCAAGTCTTTCAGTGAAAGGTCCCATCTGCGAGAGCACCAGCGAACACATACTGGCGAGAAGCCGTACAAGTGTCCCGAGTGCGGCAAGAGTTTTAGTTCCAAAAAACACCTGGCCGAACATCAGCGGACTCACACAGGGGAGAAGCCCTATAAATGCCCCGAGTGCGGCAAGAGCTTTAGCGATCCCGGGGCCCTCGTCCGACATCAGAGGACCCACACAGGGGAGAAACCTTACAAGTGTCCTGAATGCGGCAAATCTTTCAGCCAGAGAGCAAACCTGCGAGCTCACCAGAGAACCCATACTGGCGAAAAGCCTTATAAATGCCCTGAATGCGGGAAGAGTTTCAGCCGCTCTGACCACCTGACTACTCACCAGCGGACACACACTGGGAAGAAAACTAGCAGCGCTGCTGACCCCAAGAAGAAGAGGAAGGTGTCGCCAGGGATCCGTCGACTTGACGCGTTGATATCAACAAGTTTGTACAAAAAAGCAGGCTACAAAGAGGCCAGCGGTTCCGGACGGGCTGACGCATTGGACGATTTTGATCTGGATATGCTGGGAAGTGACGCCCTCGATGATTTTGACCTTGACATGCTTGGTTCGGATGCCCTTGATGACTTTGACCTCGACATGCTCGGCAGTGACGCCCTTGATGATTTCGACCTGGACATGCTGATTAACTCTAGAAGTTCCGGATCTCCGAAAAAGAAACGCAAAGTTGGTAGCCAGTACCTGCCCGACACCGACGACCGGCACCGGATCGAGGAAAAGCGGAAGCGGACCTACGAGACATTCAAGAGCATCATGAAGAAGTCCCCCTTCAGCGGCCCCACCGACCCTAGACCTCCACCTAGAAGAATCGCCGTGCCCAGCAGATCCAGCGCCAGCGTGCCAAAACCTGCCCCCCAGCCTTACCCCTTCACCAGCAGCCTGAGCACCATCAACTACGACGAGTTCCCTACCATGGTGTTCCCCAGCGGCCAGATCTCTCAGGCCTCTGCTCTGGCTCCAGCCCCTCCTCAGGTGCTGCCTCAGGCTCCTGCTCCTGCACCAGCTCCAGCCATGGTGTCTGCACTGGCTCAGGCACCAGCACCCGTGCCTGTGCTGGCTCCTGGACCTCCACAGGCTGTGGCTCCACCAGCCCCTAAACCTACACAGGCCGGCGAGGGCACACTGTCTGAAGCTCTGCTGCAGCTGCAGTTCGACGACGAGGATCTGGGAGCCCTGCTGGGAAACAGCACCGATCCTGCCGTGTTCACCGACCTGGCCAGCGTGGACAACAGCGAGTTCCAGCAGCTGCTGAACCAGGGCATCCCTGTGGCCCCTCACACCACCGAGCCCATGCTGATGGAATACCCCGAGGCCATCACCCGGCTCGTGACAGGCGCTCAGAGGCCTCCTGATCCAGCTCCTGCCCCTCTGGGAGCACCAGGCCTGCCTAATGGACTGCTGTCTGGCGACGAGGACTTCAGCTCTATCGCCGATATGGATTTCTCAGCCTTGCTGGGCTCTGGCAGCGGCAGCCGGGATTCCAGGGAAGGGATGTTTTTGCCGAAGCCTGAGGCCGGCTCCGCTATTAGTGACGTGTTTGAGGGCCGCGAGGTGTGCCAGCCAAAACGAATCCGGCCATTTCATCCTCCAGGAAGTCCATGGGCCAACCGCCCACTCCCCGCCAGCCTCGCACCAACACCAACCGGTCCAGTACATGAGCCAGTCGGGTCACTGACCCCGGCACCAGTCCCTCAGCCACTGGATCCAGCGCCCGCAGTGACTCCCGAGGCCAGTCACCTGTTGGAGGATCCCGATGAAGAGACGAGCCAGGCTGTCAAAGCCCTTCGGGAGATGGCCGATACTGTGATTCCCCAGAAGGAAGAGGCTGCAATCTGTGGCCAAATGGACCTTTCCCATCCGCCCCCAAGGGGCCATCTGGATGAGCTGACAACCACACTTGAGTCCATGACCGAGGATCTGAACCTGGACTCACCCCTGACCCCGGAATTGAACGAGATTCTGGATACCTTCCTGAACGACGAGTGCCTCTTGCATGCCATGCATATCAGCACAGGACTGTCCATCTTCGACACATCTCTGTTTSEQ ID NO: 10 (5′ LTR primer for CHIP analysis of ZFP-362-VPRbinding the 5′ LTR) TTTCCGCTGGGGACTTTCCAGSEQ ID NO: 11 (3′ LTR primer for CHIP analysis of ZFP-362-VPRbinding the 5′ LTR) ACTCAAGGCAAGCTTTATTGAGGCSEQ ID NO: 12 (ZFP-362-VPR-NLS. ZFP-362 sequence)LEPGEKPYKCPECGKSFSRKDNLKNHQRTHTGEKPYKCPECGKSFSQRAHLERHQRTHTGEKPYKCPECGKSFSERSHLREHQRTHTGEKPYKCPECGKSFSSKKHLAEHQRTHTGEKPYKCPECGKSFSDPGALVRHQRTHTGEKPYKCPECGKSFSQRANLRAHQRTHTGEKPYKCPECGKSFSRSDHLTTHQRTHTGKKTSSAADPKKKRKVSEQ ID NO: 13 (ZFP-362-VPR-NLS. VPR sequence)SPGIRRLDALISTSLYKKAGYKEASGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINSRSSGSPKKKRKVGSQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLF SEQ ID NO: 14 (ZFP-362-VPR-Tat. TAT sequence)GRKKRRQRRRVDL SEQ ID NO: 15 (ZFP-362-VPR-NLS. NLS sequence)KRPAATKKAGQAKKKK SEQ ID NO: 16 (ZFP-362-VPR-NLS. Myc sequence)EQKLISEEDLLSEQ ID NO: 17 (ZFP-362-VPR-NLS. The recombinant protein Myc-NLS-ZFP-362-VPR, is shown below)EQKLISEEDLLKRPAATKKAGQAKKKKLEPGEKPYKCPECGKSFSRKDNLKNHQRTHTGEKPYKCPECGKSFSQRAHLERHQRTHTGEKPYKCPECGKSFSERSHLREHQRTHTGEKPYKCPECGKSFSSKKHLAEHQRTHTGEKPYKCPECGKSFSDPGALVRHQRTHTGEKPYKCPECGKSFSQRANLRAHQRTHTGEKPYKCPECGKSFSRSDHLTTHQRTHTGKKTSSAADPKKKRKVSPGIRRLDALISTSLYKKAGYKEASGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINSRSSGSPKKKRKVGSQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLFSEQ ID NO: 18 (ZFP-362-VPR-Tat. The recombinant protein TAT-Myc-NLS-ZFP-362-VPR is shown below)GRKKRRQRRRVDLEQKLISEEDLLKRPAATKKAGQAKKKKLEPGEKPYKCPECGKSFSRKDNLKNHQRTHTGEKPYKCPECGKSFSQRAHLERHQRTHTGEKPYKCPECGKSFSERSHLREHQRTHTGEKPYKCPECGKSFSSKKHLAEHQRTHTGEKPYKCPECGKSFSDPGALVRHQRTHTGEKPYKCPECGKSFSQRANLRAHQRTHTGEKPYKCPECGKSFSRSDHLTTHQRTHTGKKTSSAADPKKKRKVSPGIRRLDALISTSLYKKAGYKEASGSGRADALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLINSRSSGSPKKKRKVGSQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLF

What is claimed is:
 1. A recombinant peptide comprising a zinc fingerdomain, wherein said recombinant peptide is encoded by SEQ ID NO. 3 or aderivative thereof having at least 90% nucleic acid sequence identity toSEQ ID NO. 3, and wherein said recombinant peptide binds to a targetnucleotide sequence, said target nucleotide sequence comprising thesequence of SEQ ID NO. 1 or a derivative thereof having at least 75%nucleotide sequence identity to the sequence of SEQ ID NO.
 1. 2. Therecombinant peptide of claim 1, wherein said target nucleotide sequencecomprises the sequence of SEQ ID NO. 2 or a derivative thereof having atleast 75% nucleotide sequence identity to the sequence of SEQ ID NO. 2.3. The recombinant peptide of claim 1, wherein said target nucleotidesequence is a long terminal repeat (LTR) sequence of HumanImmunodeficiency Virus (HIV).
 4. The recombinant peptide of claim 1,wherein the recombinant peptide is encoded by the sequence of SEQ ID NO.3 or a derivative thereof having at least 95% nucleic acid sequenceidentity to the sequence of SEQ ID NO.
 3. 5. The recombinant peptide ofclaim 1, wherein the recombinant peptide is encoded by the sequence ofSEQ ID NO. 3 or a derivative thereof having at least 99% nucleic acidsequence identity to the sequence of SEQ ID NO.
 3. 6. The recombinantpeptide of claim 1, wherein the recombinant peptide further comprises apeptide capable of transcriptional activation.
 7. The recombinantpeptide of claim 6, wherein said peptide capable of transcriptionalactivation is selected from the group consisting of Viral Protein R(VPR), p65 transactivating subunit of NF-kappa B, heat-shock factor 1(HSF) activation domain, VP64 (tetramer of VP16) activation domain andsynergistic activation mediator (SAM).
 8. The recombinant peptide ofclaim 1, wherein the recombinant peptide further comprises (a) one ormore nuclear localization signal (NLS) sequences, (b) a peptidecomprising a TAT peptide, or (c) a peptide comprising a cleavablemaltose binding domain.
 9. The recombinant peptide of claim 1, whereinthe recombinant peptide can cross a blood brain barrier.
 10. Anucleotide sequence encoding the recombinant peptide of claim 1, whereinthe sequence is SEQ ID NO:3 or a derivative thereof having at least 90%nucleic acid sequence identity to SEQ ID NO.
 3. 11. An expression vectorcomprising the nucleotide sequence of claim
 10. 12. The expressionvector of claim 11, wherein said nucleotide sequence is expressed underthe control of a CMV promoter, a H1 promoter or an EF1 alpha promoter.